RNA-Guided Transcriptional Regulation

ABSTRACT

Methods of modulating expression of a target nucleic acid in a cell are provided including introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to DNA, wherein the DNA includes the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding a nuclease-null Cas9 protein that binds to the DNA and is guided by the one or more RNAs, introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the one or more RNAs, the nuclease-null Cas9 protein, and the transcriptional regulator protein or domain are expressed, wherein the one or more RNAs, the nuclease-null Cas9 protein and the transcriptional regulator protein or domain co-localize to the DNA and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (010498_01255_ST25.txt;Size: 69,820 bytes; and Date of Creation: Jun. 14, 2019) is hereinincorporated by reference in its entirety.

RELATED APPLICATION DATA

This application is a continuation of U.S. patent application Ser. No.14/319,530, filed on Jun. 30, 2014, which is a continuation of PCTapplication no. PCT/US2014/040868, designating the United States andfiled Jun. 4, 2014; which claims the benefit U.S. Provisional PatentApplication No. 61/830,787 filed on Jun. 4, 2013; each of which arehereby incorporated by reference in their entireties.

STATEMENT OF GOVERNMENT INTERESTS

This invention was made with government support under Grant No. P50HG005550 from the National Institutes of health and DE-FG02-02ER63445from the Department of Energy. The government has certain rights in theinvention.

BACKGROUND

Bacterial and archaeal CRISPR-Cas systems rely on short guide RNAs incomplex with Cas proteins to direct degradation of complementarysequences present within invading foreign nucleic acid. See Deltcheva,E. et al. CRISPR RNA maturation by trans-encoded small RNA and hostfactor RNase III. Nature 471, 602-607 (2011); Gasiunas, G, Barrangou,R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complexmediates specific DNA cleavage for adaptive immunity in bacteria.Proceedings of the National Academy of Sciences of the United States ofAmerica 109, E2579-2586 (2012); Jinek, M. et al. A programmabledual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science337, 816-821 (2012); Sapranauskas, R. et al. The Streptococcusthermophilus CRISPR/Cas system provides immunity in Escherichia coli.Nucleic acids research 39, 9275-9282 (2011); and Bhaya, D., Davison, M.& Barrangou, R. CRISPR-Cas systems in bacteria and archaea: versatilesmall RNAs for adaptive defense and regulation. Annual review ofgenetics 45, 273-297 (2011). A recent in vitro reconstitution of the S.pyogenes type II CRISPR system demonstrated that crRNA (“CRISPR RNA”)fused to a normally trans-encoded tracrRNA (“trans-activating CRISPRRNA”) is sufficient to direct Cas9 protein to sequence-specificallycleave target DNA sequences matching the crRNA. Expressing a gRNAhomologous to a target site results in Cas9 recruitment and degradationof the target DNA. See H. Deveau et al., Phage response toCRISPR-encoded resistance in Streptococcus thermophilus. Journal ofBacteriology 190, 1390 (Feb, 2008).

SUMMARY

Aspects of the present disclosure are directed to a complex of a guideRNA, a DNA binding protein and a double stranded DNA target sequence.According to certain aspects, DNA binding proteins within the scope ofthe present disclosure include a protein that forms a complex with theguide RNA and with the guide RNA guiding the complex to a doublestranded DNA sequence wherein the complex binds to the DNA sequence.This aspect of the present disclosure may be referred to asco-localization of the RNA and DNA binding protein to or with the doublestranded DNA. In this manner, a DNA binding protein-guide RNA complexmay be used to localize a transcriptional regulator protein or domain attarget DNA so as to regulate expression of target DNA.

According to certain aspects, a method of modulating expression of atarget nucleic acid in a cell is provided including introducing into thecell a first foreign nucleic acid encoding one or more RNAs (ribonucleicacids) complementary to DNA (deoxyribonucleic acid), wherein the DNAincludes the target nucleic acid, introducing into the cell a secondforeign nucleic acid encoding an RNA guided nuclease-null DNA bindingprotein that binds to the DNA and is guided by the one or more RNAs,introducing into the cell a third foreign nucleic acid encoding atranscriptional regulator protein or domain, wherein the one or moreRNAs, the RNA guided nuclease-null DNA binding protein, and thetranscriptional regulator protein or domain are expressed, wherein theone or more RNAs, the RNA guided nuclease-null DNA binding protein andthe transcriptional regulator protein or domain co-localize to the DNAand wherein the transcriptional regulator protein or domain regulatesexpression of the target nucleic acid.

According to one aspect, the foreign nucleic acid encoding an RNA guidednuclease-null DNA binding protein further encodes the transcriptionalregulator protein or domain fused to the RNA guided nuclease-null DNAbinding protein. According to one aspect, the foreign nucleic acidencoding one or more RNAs further encodes a target of an RNA-bindingdomain and the foreign nucleic acid encoding the transcriptionalregulator protein or domain further encodes an RNA-binding domain fusedto the transcriptional regulator protein or domain.

According to one aspect, the cell is a eukaryotic cell. According to oneaspect, the cell is a yeast cell, a plant cell or an animal cell.According to one aspect, the cell is a mammalian cell.

According to one aspect, the RNA is between about 10 to about 500nucleotides. According to one aspect, the RNA is between about 20 toabout 100 nucleotides.

According to one aspect, the transcriptional regulator protein or domainis a transcriptional activator. According to one aspect, thetranscriptional regulator protein or domain upregulates expression ofthe target nucleic acid. According to one aspect, the transcriptionalregulator protein or domain upregulates expression of the target nucleicacid to treat a disease or detrimental condition. According to oneaspect, the target nucleic acid is associated with a disease ordetrimental condition.

According to one aspect, the one or more RNAs is a guide RNA. Accordingto one aspect, the one or more RNAs is a tracrRNA-crRNA fusion.According to one aspect, the guide RNA includes a spacer sequence and atracer mate sequence. The guide RNA may also include a tracr sequence, aportion of which hybridizes to the tracr mate sequence. The guide RNAmay also include a linker nucleic acid sequence which links the tracermate sequence and the tracr sequence to produce the tracrRNA-crRNAfusion. The spacer sequence binds to target DNA, such as byhybridization.

According to one aspect, the guide RNA includes a truncated spacersequence. According to one aspect, the guide RNA includes a truncatedspacer sequence having a 1 base truncation at the 5′ end of the spacersequence. According to one aspect, the guide RNA includes a truncatedspacer sequence having a 2 base truncation at the 5′ end of the spacersequence. According to one aspect, the guide RNA includes a truncatedspacer sequence having a 3 base truncation at the 5′ end of the spacersequence. According to one aspect, the guide RNA includes a truncatedspacer sequence having a 4 base truncation at the 5′ end of the spacersequence. Accordingly, the spacer sequence may have a 1 to 4 basetruncation at the 5′ end of the spacer sequence.

According to certain embodiments, the spacer sequence may includebetween about 16 to about 20 nucleotides which hybridize to the targetnucleic acid sequence. According to certain embodiments, the spacersequence may include about 20 nucleotides which hybridize to the targetnucleic acid sequence.

According to certain aspects, the linker nucleic acid sequence mayinclude between about 4 and about 6 nucleic acids.

According to certain aspects, the tracr sequence may include betweenabout 60 to about 500 nucleic acids. According to certain aspects, thetracr sequence may include between about 64 to about 500 nucleic acids.According to certain aspects, the tracr sequence may include betweenabout 65 to about 500 nucleic acids. According to certain aspects, thetracr sequence may include between about 66 to about 500 nucleic acids.According to certain aspects, the tracr sequence may include betweenabout 67 to about 500 nucleic acids. According to certain aspects, thetracr sequence may include between about 68 to about 500 nucleic acids.According to certain aspects, the tracr sequence may include betweenabout 69 to about 500 nucleic acids. According to certain aspects, thetracr sequence may include between about 70 to about 500 nucleic acids.According to certain aspects, the tracr sequence may include betweenabout 80 to about 500 nucleic acids. According to certain aspects, thetracr sequence may include between about 90 to about 500 nucleic acids.According to certain aspects, the tracr sequence may include betweenabout 100 to about 500 nucleic acids.

According to certain aspects, the tracr sequence may include betweenabout 60 to about 200 nucleic acids. According to certain aspects, thetracr sequence may include between about 64 to about 200 nucleic acids.According to certain aspects, the tracr sequence may include betweenabout 65 to about 200 nucleic acids. According to certain aspects, thetracr sequence may include between about 66 to about 200 nucleic acids.According to certain aspects, the tracr sequence may include betweenabout 67 to about 200 nucleic acids. According to certain aspects, thetracr sequence may include between about 68 to about 200 nucleic acids.According to certain aspects, the tracr sequence may include betweenabout 69 to about 200 nucleic acids. According to certain aspects, thetracr sequence may include between about 70 to about 200 nucleic acids.According to certain aspects, the tracr sequence may include betweenabout 80 to about 200 nucleic acids. According to certain aspects, thetracr sequence may include between about 90 to about 200 nucleic acids.According to certain aspects, the tracr sequence may include betweenabout 100 to about 200 nucleic acids.

An exemplary guide RNA is depicted in FIG. 5B.

According to one aspect, the DNA is genomic DNA, mitochondrial DNA,viral DNA, or exogenous DNA.

According to certain aspects, a method of modulating expression of atarget nucleic acid in a cell is provided including introducing into thecell a first foreign nucleic acid encoding one or more RNAs (ribonucleicacids) complementary to DNA (deoxyribonucleic acid), wherein the DNAincludes the target nucleic acid, introducing into the cell a secondforeign nucleic acid encoding an RNA guided nuclease-null DNA bindingprotein of a Type II CRISPR System that binds to the DNA and is guidedby the one or more RNAs, introducing into the cell a third foreignnucleic acid encoding a transcriptional regulator protein or domain,wherein the one or more RNAs, the RNA guided nuclease-null DNA bindingprotein of a Type II CRISPR System, and the transcriptional regulatorprotein or domain are expressed, wherein the one or more RNAs, the RNAguided nuclease-null DNA binding protein of a Type II CRISPR System andthe transcriptional regulator protein or domain co-localize to the DNAand wherein the transcriptional regulator protein or domain regulatesexpression of the target nucleic acid.

According to one aspect, the foreign nucleic acid encoding an RNA guidednuclease-null DNA binding protein of a Type II CRISPR System furtherencodes the transcriptional regulator protein or domain fused to the RNAguided nuclease-null DNA binding protein of a Type II CRISPR System.According to one aspect, the foreign nucleic acid encoding one or moreRNAs further encodes a target of an RNA-binding domain and the foreignnucleic acid encoding the transcriptional regulator protein or domainfurther encodes an RNA-binding domain fused to the transcriptionalregulator protein or domain

According to one aspect, the cell is a eukaryotic cell. According to oneaspect, the cell is a yeast cell, a plant cell or an animal cell.According to one aspect, the cell is a mammalian cell.

According to one aspect, the RNA is between about 10 to about 500nucleotides. According to one aspect, the RNA is between about 20 toabout 100 nucleotides.

According to one aspect, the transcriptional regulator protein or domainis a transcriptional activator. According to one aspect, thetranscriptional regulator protein or domain upregulates expression ofthe target nucleic acid. According to one aspect, the transcriptionalregulator protein or domain upregulates expression of the target nucleicacid to treat a disease or detrimental condition. According to oneaspect, the target nucleic acid is associated with a disease ordetrimental condition.

According to one aspect, the one or more RNAs is a guide RNA. Accordingto one aspect, the one or more RNAs is a tracrRNA-crRNA fusion.

According to one aspect, the DNA is genomic DNA, mitochondrial DNA,viral DNA, or exogenous DNA.

According to certain aspects, a method of modulating expression of atarget nucleic acid in a cell is provided including introducing into thecell a first foreign nucleic acid encoding one or more RNAs (ribonucleicacids) complementary to DNA (deoxyribonucleic acid), wherein the DNAincludes the target nucleic acid, introducing into the cell a secondforeign nucleic acid encoding a nuclease-null Cas9 protein that binds tothe DNA and is guided by the one or more RNAs, introducing into the cella third foreign nucleic acid encoding a transcriptional regulatorprotein or domain, wherein the one or more RNAs, the nuclease-null Cas9protein, and the transcriptional regulator protein or domain areexpressed, wherein the one or more RNAs, the nuclease-null Cas9 proteinand the transcriptional regulator protein or domain co-localize to theDNA and wherein the transcriptional regulator protein or domainregulates expression of the target nucleic acid.

According to one aspect, the foreign nucleic acid encoding anuclease-null Cas9 protein further encodes the transcriptional regulatorprotein or domain fused to the nuclease-null Cas9 protein.

According to one aspect, the foreign nucleic acid encoding one or moreRNAs further encodes a target of an RNA-binding domain and the foreignnucleic acid encoding the transcriptional regulator protein or domainfurther encodes an RNA-binding domain fused to the transcriptionalregulator protein or domain

According to one aspect, the cell is a eukaryotic cell. According to oneaspect, the cell is a yeast cell, a plant cell or an animal cell.According to one aspect, the cell is a mammalian cell.

According to one aspect, the RNA is between about 10 to about 500nucleotides. According to one aspect, the RNA is between about 20 toabout 100 nucleotides.

According to one aspect, the transcriptional regulator protein or domainis a transcriptional activator. According to one aspect, thetranscriptional regulator protein or domain upregulates expression ofthe target nucleic acid. According to one aspect, the transcriptionalregulator protein or domain upregulates expression of the target nucleicacid to treat a disease or detrimental condition. According to oneaspect, the target nucleic acid is associated with a disease ordetrimental condition.

According to one aspect, the one or more RNAs is a guide RNA. Accordingto one aspect, the one or more RNAs is a tracrRNA-crRNA fusion.

According to one aspect, the DNA is genomic DNA, mitochondrial DNA,viral DNA, or exogenous DNA.

According to one aspect a cell is provided that includes a first foreignnucleic acid encoding one or more RNAs complementary to DNA, wherein theDNA includes a target nucleic acid, a second foreign nucleic acidencoding an RNA guided nuclease-null DNA binding protein, and a thirdforeign nucleic acid encoding a transcriptional regulator protein ordomain wherein the one or more RNAs, the RNA guided nuclease-null DNAbinding protein and the transcriptional regulator protein or domain aremembers of a co-localization complex for the target nucleic acid.

According to one aspect, the foreign nucleic acid encoding an RNA guidednuclease-null DNA binding protein further encodes the transcriptionalregulator protein or domain fused to an RNA guided nuclease-null DNAbinding protein. According to one aspect, the foreign nucleic acidencoding one or more RNAs further encodes a target of an RNA-bindingdomain and the foreign nucleic acid encoding the transcriptionalregulator protein or domain further encodes an RNA-binding domain fusedto the transcriptional regulator protein or domain.

According to one aspect, the cell is a eukaryotic cell. According to oneaspect, the cell is a yeast cell, a plant cell or an animal cell.According to one aspect, the cell is a mammalian cell.

According to one aspect, the RNA is between about 10 to about 500nucleotides. According to one aspect, the RNA is between about 20 toabout 100 nucleotides.

According to one aspect, the transcriptional regulator protein or domainis a transcriptional activator. According to one aspect, thetranscriptional regulator protein or domain upregulates expression ofthe target nucleic acid. According to one aspect, the transcriptionalregulator protein or domain upregulates expression of the target nucleicacid to treat a disease or detrimental condition. According to oneaspect, the target nucleic acid is associated with a disease ordetrimental condition.

According to one aspect, the one or more RNAs is a guide RNA. Accordingto one aspect, the one or more RNAs is a tracrRNA-crRNA fusion.

According to one aspect, the DNA is genomic DNA, mitochondrial DNA,viral DNA, or exogenous DNA.

According to certain aspects, the RNA guided nuclease-null DNA bindingprotein is an RNA guided nuclease-null DNA binding protein of a Type IICRISPR System. According to certain aspects, the RNA guidednuclease-null DNA binding protein is a nuclease-null Cas9 protein.

According to one aspect, a method of altering a DNA target nucleic acidin a cell is provided that includes introducing into the cell a firstforeign nucleic acid encoding two or more RNAs with each RNA beingcomplementary to an adjacent site in the DNA target nucleic acid,introducing into the cell a second foreign nucleic acid encoding atleast one RNA guided DNA binding protein nickase and being guided by thetwo or more RNAs, wherein the two or more RNAs and the at least one RNAguided DNA binding protein nickase are expressed and wherein the atleast one RNA guided DNA binding protein nickase co-localizes with thetwo or more RNAs to the DNA target nucleic acid and nicks the DNA targetnucleic acid resulting in two or more adjacent nicks.

According to one aspect, a method of altering a DNA target nucleic acidin a cell is provided that includes introducing into the cell a firstforeign nucleic acid encoding two or more RNAs with each RNA beingcomplementary to an adjacent site in the DNA target nucleic acid,introducing into the cell a second foreign nucleic acid encoding atleast one RNA guided DNA binding protein nickase of a Type II CRISPRSystem and being guided by the two or more RNAs, wherein the two or moreRNAs and the at least one RNA guided DNA binding protein nickase of aType II CRISPR System are expressed and wherein the at least one RNAguided DNA binding protein nickase of a Type II CRISPR Systemco-localizes with the two or more RNAs to the DNA target nucleic acidand nicks the DNA target nucleic acid resulting in two or more adjacentnicks.

According to one aspect, a method of altering a DNA target nucleic acidin a cell is provided that includes introducing into the cell a firstforeign nucleic acid encoding two or more RNAs with each RNA beingcomplementary to an adjacent site in the DNA target nucleic acid,introducing into the cell a second foreign nucleic acid encoding atleast one Cas9 protein nickase having one inactive nuclease domain andbeing guided by the two or more RNAs, wherein the two or more RNAs andthe at least one Cas9 protein nickase are expressed and wherein the atleast one Cas9 protein nickase co-localizes with the two or more RNAs tothe DNA target nucleic acid and nicks the DNA target nucleic acidresulting in two or more adjacent nicks.

According to the methods of altering a DNA target nucleic acid, the twoor more adjacent nicks are on the same strand of the double strandedDNA. According to one aspect, the two or more adjacent nicks are on thesame strand of the double stranded DNA and result in homologousrecombination. According to one aspect, the two or more adjacent nicksare on different strands of the double stranded DNA. According to oneaspect, the two or more adjacent nicks are on different strands of thedouble stranded DNA and create double stranded breaks. According to oneaspect, the two or more adjacent nicks are on different strands of thedouble stranded DNA and create double stranded breaks resulting innonhomologous end joining. According to one aspect, the two or moreadjacent nicks are on different strands of the double stranded DNA andare offset with respect to one another. According to one aspect, the twoor more adjacent nicks are on different strands of the double strandedDNA and are offset with respect to one another and create doublestranded breaks. According to one aspect, the two or more adjacent nicksare on different strands of the double stranded DNA and are offset withrespect to one another and create double stranded breaks resulting innonhomologous end joining. According to one aspect, the method furtherincludes introducing into the cell a third foreign nucleic acid encodinga donor nucleic acid sequence wherein the two or more nicks results inhomologous recombination of the target nucleic acid with the donornucleic acid sequence.

According to one aspect, a method of altering a DNA target nucleic acidin a cell is provided including introducing into the cell a firstforeign nucleic acid encoding two or more RNAs with each RNA beingcomplementary to an adjacent site in the DNA target nucleic acid,introducing into the cell a second foreign nucleic acid encoding atleast one RNA guided DNA binding protein nickase and being guided by thetwo or more RNAs, and wherein the two or more RNAs and the at least oneRNA guided DNA binding protein nickase are expressed and wherein the atleast one RNA guided DNA binding protein nickase co-localizes with thetwo or more RNAs to the DNA target nucleic acid and nicks the DNA targetnucleic acid resulting in two or more adjacent nicks, and wherein thetwo or more adjacent nicks are on different strands of the doublestranded DNA and create double stranded breaks resulting infragmentation of the target nucleic acid thereby preventing expressionof the target nucleic acid.

According to one aspect, a method of altering a DNA target nucleic acidin a cell is provided including introducing into the cell a firstforeign nucleic acid encoding two or more RNAs with each RNA beingcomplementary to an adjacent site in the DNA target nucleic acid,introducing into the cell a second foreign nucleic acid encoding atleast one RNA guided DNA binding protein nickase of a Type II CRISPRsystem and being guided by the two or more RNAs, and wherein the two ormore RNAs and the at least one RNA guided DNA binding protein nickase ofa Type II CRISPR System are expressed and wherein the at least one RNAguided DNA binding protein nickase of a Type II CRISPR Systemco-localizes with the two or more RNAs to the DNA target nucleic acidand nicks the DNA target nucleic acid resulting in two or more adjacentnicks, and wherein the two or more adjacent nicks are on differentstrands of the double stranded DNA and create double stranded breaksresulting in fragmentation of the target nucleic acid thereby preventingexpression of the target nucleic acid.

According to one aspect, a method of altering a DNA target nucleic acidin a cell is provided including introducing into the cell a firstforeign nucleic acid encoding two or more RNAs with each RNA beingcomplementary to an adjacent site in the DNA target nucleic acid,introducing into the cell a second foreign nucleic acid encoding atleast one Cas9 protein nickase having one inactive nuclease domain andbeing guided by the two or more RNAs, and wherein the two or more RNAsand the at least one Cas9 protein nickase are expressed and wherein theat least one Cas9 protein nickase co-localizes with the two or more RNAsto the DNA target nucleic acid and nicks the DNA target nucleic acidresulting in two or more adjacent nicks, and wherein the two or moreadjacent nicks are on different strands of the double stranded DNA andcreate double stranded breaks resulting in fragmentation of the targetnucleic acid thereby preventing expression of the target nucleic acid.

According to one aspect, a cell is provided including a first foreignnucleic acid encoding two or more RNAs with each RNA being complementaryto an adjacent site in a DNA target nucleic acid, and a second foreignnucleic acid encoding at least one RNA guided DNA binding proteinnickase, and wherein the two or more RNAs and the at least one RNAguided DNA binding protein nickase are members of a co-localizationcomplex for the DNA target nucleic acid.

According to one aspect, the RNA guided DNA binding protein nickase isan RNA guided DNA binding protein nickase of a Type II CRISPR System.According to one aspect, the RNA guided DNA binding protein nickase is aCas9 protein nickase having one inactive nuclease domain

According to one aspect, the cell is a eukaryotic cell. According to oneaspect, the cell is a yeast cell, a plant cell or an animal cell.According to one aspect, the cell is a mammalian cell.

According to one aspect, the RNA includes between about 10 to about 500nucleotides. According to one aspect, the RNA includes between about 20to about 100 nucleotides.

According to one aspect, the target nucleic acid is associated with adisease or detrimental condition.

According to one aspect, the two or more RNAs are guide RNAs. Accordingto one aspect, the two or more RNAs are tracrRNA-crRNA fusions.

According to one aspect, the DNA target nucleic acid_is genomic DNA,mitochondrial DNA, viral DNA, or exogenous DNA.

Further features and advantages of certain embodiments of the presentinvention will become more fully apparent in the following descriptionof embodiments and drawings thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains drawings executed in color.Copies of this patent or patent application publication with the colordrawings will be provided by the Office upon request and payment of thenecessary fee. The foregoing and other features and advantages of thepresent embodiments will be more fully understood from the followingdetailed description of illustrative embodiments taken in conjunctionwith the accompanying drawings in which:

FIG. 1A and FIG. 1B are schematics of RNA-guided transcriptionalactivation. FIG. 1C is a design of a reporter construct (SEQ ID Nos:62and 63). FIGS. 1D-1 and 1D-2 show data demonstrating that Cas9N-VP64fusions display RNA-guided transcriptional activation as assayed by bothfluorescence-activated cell sorting (FACS) and immunofluorescence assays(IF). FIGS. 1E-1 and 1E-2 show assay data by FACS and IF demonstratinggRNA sequence-specific transcriptional activation from reporterconstructs in the presence of Cas9N, MS2-VP64 and gRNA bearing theappropriate MS2 aptamer binding sites. FIG. 1F depicts datademonstrating transcriptional induction by individual gRNAs and multiplegRNAs.

FIG. 2A depicts a methodology for evaluating the landscape of targetingby Cas9-gRNA complexes and TALEs. FIG. 2B depicts data demonstratingthat a Cas9-gRNA complex is on average tolerant to 1-3 mutations in itstarget sequences. FIG. 2C depicts data demonstrating that the Cas9-gRNAcomplex is largely insensitive to point mutations, except thoselocalized to the PAM sequence. FIG. 2D depicts heat plot datademonstrating that introduction of 2 base mismatches significantlyimpairs the Cas9-gRNA complex activity. FIG. 2E depicts datademonstrating that an 18-mer TALE reveals is on average tolerant to 1-2mutations in its target sequence. FIG. 2F depicts data demonstrating the18-mer TALE is, similar to the Cas9-gRNA complexes, largely insensitiveto single base mismatched in its target. FIG. 2G depicts heat plot datademonstrating that introduction of 2 base mismatches significantlyimpairs the 18-mer TALE activity.

FIG. 3A depicts a schematic of a guide RNA design. FIG. 3B depicts datashowing percentage rate of non-homologous end joining for off-set nicksleading to 5′ overhangs and off-set nicks leading to 3′ overhangs. FIG.3C depicts data showing percentage rate of targeting for off-set nicksleading to 5′ overhangs and off-set nicks leading to 3′ overhangs.

FIG. 4A is a schematic of a metal coordinating residue in RuvC PDB ID:4EP4 (blue) position D7 (left), a schematic of HNH endonuclease domainsfrom PDB IDs: 3M7K (orange) and 4H9D (cyan) including a coordinatedMg-ion (gray sphere) and DNA from 3M7K (purple) (middle) and a list ofmutants analyzed (right). FIG. 4B depicts data showing undetectablenuclease activity for Cas9 mutants m3 and m4, and also their respectivefusions with VP64. FIG. 4C is a higher-resolution examination of thedata in FIG. 4B.

FIG. 5A is a schematic of a homologous recombination assay to determineCas9-gRNA activity(SEQ ID No:64). FIGS. 5B-1 and 5B-2 depict guide RNAswith random sequence insertions and percentage rate of homologousrecombination (SEQ ID Nos:65 and 66).

FIG. 6A is a schematic of guide RNAs for the OCT4 gene. FIG. 6B depictstranscriptional activation for a promoter-luciferase reporter construct.FIG. 6C depicts transcriptional activation via qPCR of endogenous genes.

FIG. 7A is a schematic of guide RNAs for the REX1 gene. FIG. 7B depictstranscriptional activation for a promoter-luciferase reporter construct.FIG. 7C depicts transcriptional activation via qPCR of endogenous genes.

FIG. 8A depicts in schematic a high level specificity analysisprocessing flow for calculation of normalized expression levels. FIG. 8Bdepicts data of distributions of percentages of binding sites by numbersof mismatches generated within a biased construct library. Left:Theoretical distribution. Right: Distribution observed from an actualTALE construct library. FIG. 8C depicts data of distributions ofpercentages of tag counts aggregated to binding sites by numbers ofmismatches. Left: Distribution observed from the positive controlsample. Right: Distribution observed from a sample in which anon-control TALE was induced.

FIG. 9A depicts data for analysis of the targeting landscape of aCas9-gRNA complex showing tolerance to 1-3 mutations in its targetsequence. FIG. 9B depicts data for analysis of the targeting landscapeof a Cas9-gRNA complex showing insensitivity to point mutations, exceptthose localized to the PAM sequence. FIG. 9C depicts heat plot data foranalysis of the targeting landscape of a Cas9-gRNA complex showing thatintroduction of 2 base mismatches significantly impairs activity. FIG.9D depicts data from a nuclease mediated HR assay confirming that thepredicted PAM for the S. pyogenes Cas9 is NGG and also NAG (SEQ IDNos:67-69).

FIGS. 10A-1 and 10A-2 depict data from a nuclease mediated HR assayconfirming that 18-mer TALEs tolerate multiple mutations in their targetsequences (SEQ ID Nos:70-73). FIG. 10B depicts data from analysis of thetargeting landscape of TALEs of 3 different sizes (18-mer, 14-mer and10-mer). FIG. 10C depicts data for 10-mer TALEs show near single-basemismatch resolution. FIG. 10D depicts heat plot data for 10-mer TALEsshow near single-base mismatch resolution.

FIG. 11A depicts designed guide RNAs. FIG. 11B depicts percentage rateof non-homologous end joining for various guide RNAs (SEQ ID Nos:74-87).

FIGS. 12A-12B depict two genes, SOX2 and NANOG, that were regulated viasgRNAs targeting within an upstream ˜1 kb stretch of promoter DNA.

FIGS. 13A-13F depict the targeting landscape of two additional Cas9-gRNAcomplexes that were analyzed.

FIG. 14A depicts the specificity profile of two gRNAs (wild-type (SEQ IDNO:88) and mutants (SEQ ID NOs:89-90). Sequence differences arehighlighted in red. FIGS. 14B and 14C depict that this assay wasspecific for the gRNA being evaluated (data re-plotted from FIG. 13D).

FIGS. 15A-15D depict gRNA2 (FIG. 15A-B) and gRNA3 (FIG. 15C-D) bearingsingle or double-base mismatches (highlighted in red) in the spacersequence versus the target. Sequences are set forth as SEQ IDNOs:91-131.

FIGS. 16A-16D depict a nuclease assay of two independent gRNA that weretested: gRNA1 (FIGS. 16A-B) and gRNA3 (FIGS. 16C-D) bearing truncationsat the 5′ end of their spacer. Sequences are set forth as SEQ ID NOs:66,185-186 and 133-140.

FIGS. 17A-17B depict a nuclease mediated HR assay that shows the PAM forthe S. pyogenes Cas9 is NGG and also NAG. Sequences are set forth as SEQID NOs:67-69 and 141.

FIGS. 18A-18B depict a nuclease mediated HR assay that confirmed that18-mer TALEs tolerate multiple mutations in their target sequences.Sequences are set forth as SEQ ID NOs:70-73.

FIGS. 19A-19C depict a comparison of TALE monomer specificity versusTALE protein specificity. Sequences are set forth as SEQ ID NOs:142-150.

FIGS. 20A-20B depict data related to off-set nicking. Sequences are setforth as SEQ ID NOs:151-158.

FIGS. 21A-21C depict off-set nicking and NHEJ profiles. Sequences areset forth as SEQ ID NOs:159-184 and 187.

DETAILED DESCRIPTION

Embodiments of the present disclosure are based on the use of DNAbinding proteins to co-localize transcriptional regulator proteins ordomains to DNA in a manner to regulate a target nucleic acid. Such DNAbinding proteins are readily known to those of skill in the art to bindto DNA for various purposes. Such DNA binding proteins may be naturallyoccurring. DNA binding proteins included within the scope of the presentdisclosure include those which may be guided by RNA, referred to hereinas guide RNA. According to this aspect, the guide RNA and the RNA guidedDNA binding protein form a co-localization complex at the DNA. Accordingto certain aspects, the DNA binding protein may be a nuclease-null DNAbinding protein. According to this aspect, the nuclease-null DNA bindingprotein may result from the alteration or modification of a DNA bindingprotein having nuclease activity. Such DNA binding proteins havingnuclease activity are known to those of skill in the art, and includenaturally occurring DNA binding proteins having nuclease activity, suchas Cas9 proteins present, for example, in Type II CRISPR systems. SuchCas9 proteins and Type II CRISPR systems are well documented in the art.See Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 2011,pp. 467-477 including all supplementary information hereby incorporatedby reference in its entirety.

Exemplary DNA binding proteins having nuclease activity function to nickor cut double stranded DNA. Such nuclease activity may result from theDNA binding protein having one or more polypeptide sequences exhibitingnuclease activity. Such exemplary DNA binding proteins may have twoseparate nuclease domains with each domain responsible for cutting ornicking a particular strand of the double stranded DNA. Exemplarypolypeptide sequences having nuclease activity known to those of skillin the art include the McrA-HNH nuclease related domain and theRuvC-like nuclease domain Accordingly, exemplary DNA binding proteinsare those that in nature contain one or more of the McrA-HNH nucleaserelated domain and the RuvC-like nuclease domain. According to certainaspects, the DNA binding protein is altered or otherwise modified toinactivate the nuclease activity. Such alteration or modificationincludes altering one or more amino acids to inactivate the nucleaseactivity or the nuclease domain Such modification includes removing thepolypeptide sequence or polypeptide sequences exhibiting nucleaseactivity, i.e. the nuclease domain, such that the polypeptide sequenceor polypeptide sequences exhibiting nuclease activity, i.e. nucleasedomain, are absent from the DNA binding protein. Other modifications toinactivate nuclease activity will be readily apparent to one of skill inthe art based on the present disclosure. Accordingly, a nuclease-nullDNA binding protein includes polypeptide sequences modified toinactivate nuclease activity or removal of a polypeptide sequence orsequences to inactivate nuclease activity. The nuclease-null DNA bindingprotein retains the ability to bind to DNA even though the nucleaseactivity has been inactivated. Accordingly, the DNA binding proteinincludes the polypeptide sequence or sequences required for DNA bindingbut may lack the one or more or all of the nuclease sequences exhibitingnuclease activity. Accordingly, the DNA binding protein includes thepolypeptide sequence or sequences required for DNA binding but may haveone or more or all of the nuclease sequences exhibiting nucleaseactivity inactivated.

According to one aspect, a DNA binding protein having two or morenuclease domains may be modified or altered to inactivate all but one ofthe nuclease domains. Such a modified or altered DNA binding protein isreferred to as a DNA binding protein nickase, to the extent that the DNAbinding protein cuts or nicks only one strand of double stranded DNA.When guided by RNA to DNA, the DNA binding protein nickase is referredto as an RNA guided DNA binding protein nickase.

An exemplary DNA binding protein is an RNA guided DNA binding protein ofa Type II CRISPR System which lacks nuclease activity. An exemplary DNAbinding protein is a nuclease-null Cas9 protein. An exemplary DNAbinding protein is a Cas9 protein nickase.

In S. pyogenes, Cas9 generates a blunt-ended double-stranded break 3 bpupstream of the protospacer-adjacent motif (PAM) via a process mediatedby two catalytic domains in the protein: an HNH domain that cleaves thecomplementary strand of the DNA and a RuvC-like domain that cleaves thenon-complementary strand. See Jinke et al., Science 337, 816-821 (2012)hereby incorporated by reference in its entirety. Cas9 proteins areknown to exist in many Type II CRISPR systems including the following asidentified in the supplementary information to Makarova et al., NatureReviews, Microbiology, Vol. 9, June 2011, pp. 467-477: Methanococcusmaripaludis C7; Corynebacterium diphtheriae; Corynebacterium efficiensYS-314; Corynebacterium glutamicum ATCC 13032 Kitasato; Corynebacteriumglutamicum ATCC 13032 Bielefeld; Corynebacterium glutamicum R;Corynebacterium kroppenstedtii DSM 44385; Mycobacterium abscessus ATCC19977; Nocardia farcinica IFM10152; Rhodococcus erythropolis PR4;Rhodococcus jostii RHA1; Rhodococcus opacus B4 uid36573; Acidothermuscellulolyticus 11B; Arthrobacter chlorophenolicus A6; Kribbella flavidaDSM 17836 uid43465; Thermomonospora curvata DSM 43183; Bifidobacteriumdentium Bd1; Bifidobacterium longum DJO10A; Slackia heliotrinireducensDSM 20476; Persephonella marina EX H1; Bacteroides fragilis NCTC 9434;Capnocytophaga ochracea DSM 7271; Flavobacterium psychrophilum JIP02 86;Akkermansia muciniphila ATCC BAA 835; Roseiflexus castenholzii DSM13941; Roseiflexus RS1; Synechocystis PCC6803; Elusimicrobium minutumPei191; uncultured Termite group 1 bacterium phylotype Rs D17;Fibrobacter succinogenes S85; Bacillus cereus ATCC 10987; Listeriainnocua; Lactobacillus casei; Lactobacillus rhamnosus GG; Lactobacillussalivarius UCC118; Streptococcus agalactiae A909; Streptococcusagalactiae NEM316; Streptococcus agalactiae 2603; Streptococcusdysgalactiae equisimilis GGS 124; Streptococcus equi zooepidemicusMGCS10565; Streptococcus gallolyticus UCN34 uid46061; Streptococcusgordonii Challis subst CH1; Streptococcus mutans NN2025 uid46353;Streptococcus mutans; Streptococcus pyogenes M1 GAS; Streptococcuspyogenes MGAS5005; Streptococcus pyogenes MGAS2096; Streptococcuspyogenes MGAS9429; Streptococcus pyogenes MGAS10270; Streptococcuspyogenes MGAS6180; Streptococcus pyogenes MGAS315; Streptococcuspyogenes SSI-1; Streptococcus pyogenes MGAS10750; Streptococcus pyogenesNZ131; Streptococcus thermophiles CNRZ1066; Streptococcus thermophilesLMD-9; Streptococcus thermophiles LMG 18311; Clostridium botulinum A3Loch Maree; Clostridium botulinum B Eklund 17B; Clostridium botulinumBa4 657; Clostridium botulinum F Langeland; Clostridium cellulolyticumH10; Finegoldia magna ATCC 29328; Eubacterium rectale ATCC 33656;Mycoplasma gallisepticum; Mycoplasma mobile 163K; Mycoplasma penetrans;Mycoplasma synoviae 53; Streptobacillus moniliformis DSM 12112;Bradyrhizobium BTAi1; Nitrobacter hamburgensis X14; Rhodopseudomonaspalustris BisB18; Rhodopseudomonas palustris BisB5; Parvibaculumlavamentivorans DS-1; Dinoroseobacter shibae DFL 12; Gluconacetobacterdiazotrophicus Pal 5 FAPERJ; Gluconacetobacter diazotrophicus Pal 5 JGI;Azospirillum B510 uid46085; Rhodospirillum rubrum ATCC 11170;Diaphorobacter TPSY uid29975; Verminephrobacter eiseniae EF01-2;Neisseria meningitides 053442; Neisseria meningitides alpha14; Neisseriameningitides Z2491; Desulfovibrio salexigens DSM 2638; Campylobacterjejuni doylei 269 97; Campylobacter jejuni 81116; Campylobacter jejuni;Campylobacter lari RM2100; Helicobacter hepaticus; Wolinellasuccinogenes; Tolumonas auensis DSM 9187; Pseudoalteromonas atlanticaT6c; Shewanella pealeana ATCC 700345; Legionella pneumophila Paris;Actinobacillus succinogenes 130Z; Pasteurella multocida; Francisellatularensis novicida U112; Francisella tularensis holarctica; Francisellatularensis FSC 198; Francisella tularensis tularensis; Francisellatularensis WY96-3418; and Treponema denticola ATCC 35405. Accordingly,aspects of the present disclosure are directed to a Cas9 protein presentin a Type II CRISPR system, which has been rendered nuclease null orwhich has been rendered a nickase as described herein.

The Cas9 protein may be referred by one of skill in the art in theliterature as Csn1. The S. pyogenes Cas9 protein sequence that is thesubject of experiments described herein is shown below. See Deltcheva etal., Nature 471, 602-607 (2011) hereby incorporated by reference in itsentirety.

(SEQ ID NO: 1)

According to certain aspects of methods of RNA-guided genome regulationdescribed herein, Cas9 is altered to reduce, substantially reduce oreliminate nuclease activity. According to one aspect, Cas9 nucleaseactivity is reduced, substantially reduced or eliminated by altering theRuvC nuclease domain or the HNH nuclease domain. According to oneaspect, the RuvC nuclease domain is inactivated. According to oneaspect, the HNH nuclease domain is inactivated. According to one aspect,the RuvC nuclease domain and the HNH nuclease domain are inactivated.According to an additional aspect, Cas9 proteins are provided where theRuvC nuclease domain and the HNH nuclease domain are inactivated.According to an additional aspect, nuclease-null Cas9 proteins areprovided insofar as the RuvC nuclease domain and the HNH nuclease domainare inactivated. According to an additional aspect, a Cas9 nickase isprovided where either the RuvC nuclease domain or the HNH nucleasedomain is inactivated, thereby leaving the remaining nuclease domainactive for nuclease activity. In this manner, only one strand of thedouble stranded DNA is cut or nicked.

According to an additional aspect, nuclease-null Cas9 proteins areprovided where one or more amino acids in Cas9 are altered or otherwiseremoved to provide nuclease-null Cas9 proteins. According to one aspect,the amino acids include D10 and H840. See Jinke et al., Science 337,816-821 (2012). According to an additional aspect, the amino acidsinclude D839 and N863. According to one aspect, one or more or all ofD10, H840, D839 and H863 are substituted with an amino acid whichreduces, substantially eliminates or eliminates nuclease activity.According to one aspect, one or more or all of D10, H840, D839 and H863are substituted with alanine. According to one aspect, a Cas9 proteinhaving one or more or all of D10, H840, D839 and H863 substituted withan amino acid which reduces, substantially eliminates or eliminatesnuclease activity, such as alanine, is referred to as a nuclease-nullCas9 or Cas9N and exhibits reduced or eliminated nuclease activity, ornuclease activity is absent or substantially absent within levels ofdetection. According to this aspect, nuclease activity for a Cas9N maybe undetectable using known assays, i.e. below the level of detection ofknown assays.

According to one aspect, the nuclease null Cas9 protein includeshomologs and orthologs thereof which retain the ability of the proteinto bind to the DNA and be guided by the RNA. According to one aspect,the nuclease null Cas9 protein includes the sequence as set forth fornaturally occurring Cas9 from S. pyogenes and having one or more or allof D10, H840, D839 and H863 substituted with alanine and proteinsequences having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or99% homology thereto and being a DNA binding protein, such as an RNAguided DNA binding protein.

According to one aspect, the nuclease null Cas9 protein includes thesequence as set forth for naturally occurring Cas9 from S. pyogenesexcepting the protein sequence of the RuvC nuclease domain and the HNHnuclease domain and also protein sequences having at least 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% homology thereto and being aDNA binding protein, such as an RNA guided DNA binding protein. In thismanner, aspects of the present disclosure include the protein sequenceresponsible for DNA binding, for example, for co-localizing with guideRNA and binding to DNA and protein sequences homologous thereto, andneed not include the protein sequences for the RuvC nuclease domain andthe HNH nuclease domain (to the extent not needed for DNA binding), asthese domains may be either inactivated or removed from the proteinsequence of the naturally occurring Cas9 protein to produce a nucleasenull Cas9 protein.

For purposes of the present disclosure, FIG. 4A depicts metalcoordinating residues in known protein structures with homology to Cas9.Residues are labeled based on position in Cas9 sequence. Left: RuvCstructure, PDB ID: 4EP4 (blue) position D7, which corresponds to D10 inthe Cas9 sequence, is highlighted in a Mg-ion coordinating position.Middle: Structures of HNH endonuclease domains from PDB IDs: 3M7K(orange) and 4H9D (cyan) including a coordinated Mg-ion (gray sphere)and DNA from 3M7K (purple). Residues D92 and N113 in 3M7K and 4H9Dpositions D53 and N77, which have sequence homology to Cas9 amino acidsD839 and N863, are shown as sticks. Right: List of mutants made andanalyzed for nuclease activity: Cas9 wildtype; Cas9_(m1) whichsubstitutes alanine for D10; Cas9_(m2) which substitutes alanine for D10and alanine for H840; Cas9_(m3) which substitutes alanine for D10,alanine for H840, and alanine for D839; and Cas9_(m4) which substitutesalanine for D10, alanine for H840, alanine for D839, and alanine forN863.

As shown in FIG. 4B, the Cas9 mutants: m3 and m4, and also theirrespective fusions with VP64 showed undetectable nuclease activity upondeep sequencing at targeted loci. The plots show the mutation frequencyversus genomic position, with the red lines demarcating the gRNA target.FIG. 4C is a higher-resolution examination of the data in FIG. 4B andconfirms that the mutation landscape shows comparable profile asunmodified loci.

According to one aspect, an engineered Cas9-gRNA system is providedwhich enables RNA-guided genome regulation in human cells by tetheringtranscriptional activation domains to either a nuclease-null Cas9 or toguide RNAs. According to one aspect of the present disclosure, one ormore transcriptional regulatory proteins or domains (such terms are usedinterchangeably) are joined or otherwise connected to anuclease-deficient Cas9 or one or more guide RNA (gRNA). Thetranscriptional regulatory domains correspond to targeted loci.Accordingly, aspects of the present disclosure include methods andmaterials for localizing transcriptional regulatory domains to targetedloci by fusing, connecting or joining such domains to either Cas9N or tothe gRNA.

According to one aspect, a Cas9N-fusion protein capable oftranscriptional activation is provided. According to one aspect, a VP64activation domain (see Zhang et al., Nature Biotechnology 29, 149-153(2011) hereby incorporated by reference in its entirety) is joined,fused, connected or otherwise tethered to the C terminus of Cas9N.According to one method, the transcriptional regulatory domain isprovided to the site of target genomic DNA by the Cas9N protein.According to one method, a Cas9N fused to a transcriptional regulatorydomain is provided within a cell along with one or more guide RNAs. TheCas9N with the transcriptional regulatory domain fused thereto bind ator near target genomic DNA. The one or more guide RNAs bind at or neartarget genomic DNA. The transcriptional regulatory domain regulatesexpression of the target gene. According to a specific aspect, aCas9N-VP64 fusion activated transcription of reporter constructs whencombined with gRNAs targeting sequences near the promoter, therebydisplaying RNA-guided transcriptional activation.

According to one aspect, a gRNA-fusion protein capable oftranscriptional activation is provided. According to one aspect, a VP64activation domain is joined, fused, connected or otherwise tethered tothe gRNA. According to one method, the transcriptional regulatory domainis provided to the site of target genomic DNA by the gRNA. According toone method, a gRNA fused to a transcriptional regulatory domain isprovided within a cell along with a Cas9N protein. The Cas9N binds at ornear target genomic DNA. The one or more guide RNAs with thetranscriptional regulatory protein or domain fused thereto bind at ornear target genomic DNA. The transcriptional regulatory domain regulatesexpression of the target gene. According to a specific aspect, a Cas9Nprotein and a gRNA fused with a transcriptional regulatory domainactivated transcription of reporter constructs, thereby displayingRNA-guided transcriptional activation.

The gRNA tethers capable of transcriptional regulation were constructedby identifying which regions of the gRNA will tolerate modifications byinserting random sequences into the gRNA and assaying for Cas9 function.gRNAs bearing random sequence insertions at either the 5′ end of thecrRNA portion or the 3′ end of the tracrRNA portion of a chimeric gRNAretain functionality, while insertions into the tracrRNA scaffoldportion of the chimeric gRNA result in loss of function. See FIG. 5A-Bsummarizing gRNA flexibility to random base insertions. FIG. 5A is aschematic of a homologous recombination (HR) assay to determineCas9-gRNA activity. As shown in FIGS. 5B-1 and 5B-2, gRNAs bearingrandom sequence insertions at either the 5′ end of the crRNA portion orthe 3′ end of the tracrRNA portion of a chimeric gRNA retainfunctionality, while insertions into the tracrRNA scaffold portion ofthe chimeric gRNA result in loss of function. The points of insertion inthe gRNA sequence are indicated by red nucleotides. Without wishing tobe bound by scientific theory, the increased activity upon random baseinsertions at the 5′ end may be due to increased half-life of the longergRNA.

To attach VP64 to the gRNA, two copies of the MS2 bacteriophagecoat-protein binding RNA stem-loop were appended to the 3′ end of thegRNA. See Fusco et at, Current Biology: CB13, 161-167 (2003) herebyincorporated by reference in its entirety. These chimeric gRNAs wereexpressed together with Cas9N and MS2-VP64 fusion protein.Sequence-specific transcriptional activation from reporter constructswas observed in the presence of all 3 components.

FIG. 1A is a schematic of RNA-guided transcriptional activation. Asshown in FIG. 1A, to generate a Cas9N-fusion protein capable oftranscriptional activation, the VP64 activation domain was directlytethered to the C terminus of Cas9N. As shown in FIG. 1B, to generategRNA tethers capable of transcriptional activation, two copies of theMS2 bacteriophage coat-protein binding RNA stem-loop were appended tothe 3′ end of the gRNA. These chimeric gRNAs were expressed togetherwith Cas9N and MS2-VP64 fusion protein. FIG. 1C shows design of reporterconstructs used to assay transcriptional activation. The two reportersbear distinct gRNA target sites, and share a control TALE-TF targetsite. As shown in FIGS. 1D-1 and 1D-2, Cas9N-VP64 fusions displayRNA-guided transcriptional activation as assayed by bothfluorescence-activated cell sorting (FACS) and immunofluorescence assays(IF). Specifically, while the control TALE-TF activated both reporters,the Cas9N-VP64 fusion activates reporters in a gRNA sequence specificmanner As shown in FIGS. 1E-1 and 1E-2, gRNA sequence-specifictranscriptional activation from reporter constructs only in the presenceof all 3 components: Cas9N, MS2-VP64 and gRNA bearing the appropriateMS2 aptamer binding sites was observed by both FACS and IF.

According to certain aspects, methods are provided for regulatingendogenous genes using Cas9N, one or more gRNAs and a transcriptionalregulatory protein or domain According to one aspect, an endogenous genecan be any desired gene, refered to herein as a target gene. Accordingto one exemplary aspect, genes target for regulation included ZFP42(REX1) and POU5F1 (OCT4), which are both tightly regulated genesinvolved in maintenance of pluripotency. As shown in FIG. 1F, 10 gRNAstargeting a ˜5 kb stretch of DNA upstream of the transcription startsite (DNase hypersensitive sites are highlighted in green) were designedfor the REX1 gene. Transcriptional activation was assayed using either apromoter-luciferase reporter construct (see Takahashi et al., Cell 131861-872 (2007) hereby incorporated by reference in its entirety) ordirectly via qPCR of the endogenous genes.

FIG. 6A-C is directed to RNA-guided OCT4 regulation using Cas9N-VP64. Asshown in FIG. 6A, 21 gRNAs targeting a ˜5 kb stretch of DNA upstream ofthe transcription start site were designed for the OCT4 gene. The DNasehypersensitive sites are highlighted in green. FIG. 6B showstranscriptional activation using a promoter-luciferase reporterconstruct. FIG. 6C shows transcriptional activation directly via qPCR ofthe endogenous genes. While introduction of individual gRNAs modestlystimulated transcription, multiple gRNAs acted synergistically tostimulate robust multi-fold transcriptional activation.

FIG. 7A-C is directed to RNA-guided REX1 regulation using Cas9N,MS2-VP64 and gRNA+2X-MS2 aptamers. As shown in FIG. 7A, 10 gRNAstargeting a ˜5 kb stretch of DNA upstream of the transcription startsite were designed for the REX1 gene. The DNase hypersensitive sites arehighlighted in green. FIG. 7B shows transcriptional activation using apromoter-luciferase reporter construct. FIG. 7C shows transcriptionalactivation directly via qPCR of the endogenous genes. While introductionof individual gRNAs modestly stimulated transcription, multiple gRNAsacted synergistically to stimulate robust multi-fold transcriptionalactivation. In one aspect, the absence of the 2X-MS2 aptamers on thegRNA does not result in transcriptional activation. See Maeder et al.,Nature Methods 10, 243-245 (2013) and Perez-Pinera et al., NatureMethods 10, 239-242 (2013) each of which are hereby incorporated byreference in its entirety.

Accordingly, methods are directed to the use of multiple guide RNAs witha Cas9N protein and a transcriptional regulatory protein or domain toregulate expression of a target gene.

Both the Cas9 and gRNA tethering approaches were effective, with theformer displaying ˜1.5-2 fold higher potency. This difference is likelydue to the requirement for 2-component as opposed to 3-component complexassembly. However, the gRNA tethering approach in principle enablesdifferent effector domains to be recruited by distinct gRNAs so long aseach gRNA uses a different RNA-protein interaction pair. SeeKaryer-Bibens et al., Biology of the Cell/Under the Auspices o theEuropean Cell Biology Organization 100, 125-138 (2008) herebyincorporated by reference in its entirety. According to one aspect ofthe present disclosure, different target genes may be regulated usingspecific guide RNA and a generic Cas9N protein, i.e. the same or asimilar Cas9N protein for different target genes. According to oneaspect, methods of multiplex gene regulation are provided using the sameor similar Cas9N.

Methods of the present disclosure are also directed to editing targetgenes using the Cas9N proteins and guide RNAs described herein toprovide multiplex genetic and epigenetic engineering of human cells.With Cas9-gRNA targeting being an issue (see Jiang et al., NatureBiotechnology 31, 233-239 (2013) hereby incorporated by reference in itsentirety), methods are provided for in-depth interrogation of Cas9affinity for a very large space of target sequence variations.Accordingly, aspects of the present disclosure provide directhigh-throughput readout of Cas9 targeting in human cells, while avoidingcomplications introduced by dsDNA cut toxicity and mutagenic repairincurred by specificity testing with native nuclease-active Cas9.

Further aspects of the present disclosure are directed to the use of DNAbinding proteins or systems in general for the transcriptionalregulation of a target gene. One of skill in the art will readilyidentify exemplary DNA binding systems based on the present disclosure.Such DNA binding systems need not have any nuclease activity, as withthe naturally occurring Cas9 protein. Accordingly, such DNA bindingsystems need not have nuclease activity inactivated. One exemplary DNAbinding system is TALE. As a genome editing tool, usually TALE-FokIdimers are used, and for genome regulation TAEL-VP64 fusions have beenshown to be highly effective. According to one aspect, TALE specificitywas evaluated using the methodology shown in FIG. 2A. A constructlibrary in which each element of the library comprises a minimalpromoter driving a dTomato fluorescent protein is designed. Downstreamof the transcription start site m, a 24 bp (A/C/G) random transcript tagis inserted, while two TF binding sites are placed upstream of thepromoter: one is a constant DNA sequence shared by all library elements,and the second is a variable feature that bears a ‘biased’ library ofbinding sites which are engineered to span a large collection ofsequences that present many combinations of mutations away from thetarget sequence the programmable DNA targeting complex was designed tobind. This is achieved using degenerate oligonucleotides engineered tobear nucleotide frequencies at each position such that the targetsequence nucleotide appears at a 79% frequency and each other nucleotideoccurs at 7% frequency. See Patwardhan et al., Nature Biotechnology 30,265-270 (2012) hereby incorporated by reference in its entirety. Thereporter library is then sequenced to reveal the associations betweenthe 24 bp dTomato transcript tags and their corresponding ‘biased’target site in the library element. The large diversity of thetranscript tags assures that sharing of tags between different targetswill be extremely rare, while the biased construction of the targetsequences means that sites with few mutations will be associated withmore tags than sites with more mutations. Next, transcription of thedTomato reporter genes is stimulated with either a control-TF engineeredto bind the shared DNA site, or the target-TF that was engineered tobind the target site. The abundance of each expressed transcript tag ismeasured in each sample by conducting RNAseq on the stimulated cells,which is then mapped back to their corresponding binding sites using theassociation table established earlier. The control-TF is expected toexcite all library members equally since its binding site is sharedacross all library elements, while the target-TF is expected to skew thedistribution of the expressed members to those that are preferentiallytargeted by it. This assumption is used in step 5 to compute anormalized expression level for each binding site by dividing the tagcounts obtained for the target-TF by those obtained for the control-TF.

As shown in FIG. 2B, the targeting landscape of a Cas9-gRNA complexreveals that it is on average tolerant to 1-3 mutations in its targetsequences. As shown in FIG. 2C, the Cas9-gRNA complex is also largelyinsensitive to point mutations, except those localized to the PAMsequence. Notably this data reveals that the predicted PAM for the S.pyogenes Cas9 is not just NGG but also NAG. As shown in FIG. 2D,introduction of 2 base mismatches significantly impairs the Cas9-gRNAcomplex activity, however only when these are localized to the 8-10bases nearer the 3′ end of the gRNA target sequence (in the heat plotthe target sequence positions are labeled from 1-23 starting from the 5′end).

The mutational tolerance of another widely used genome editing tool,TALE domains, was determined using the transcriptional specificity assaydescribed herein. As shown in FIG. 2E, the TALE off-targeting data foran 18-mer TALE reveals that it can tolerate on average 1-2 mutations inits target sequence, and fails to activate a large majority of 3 basemismatch variants in its targets. As shown in FIG. 2F, the 18-mer TALEis, similar to the Cas9-gRNA complexes, largely insensitive to singlebase mismatched in its target. As shown in FIG. 2G, introduction of 2base mismatches significantly impairs the 18-mer TALE activity. TALEactivity is more sensitive to mismatches nearer the 5′ end of its targetsequence (in the heat plot the target sequence positions are labeledfrom 1-18 starting from the 5′ end).

Results were confirmed using targeted experiments in a nuclease assaywhich is the subject of FIGS. 10A-C directed to evaluating the landscapeof targeting by TALEs of different sizes. As shown in FIGS. 10A-1 and10A-2, using a nuclease mediated HR assay, it was confirmed that 18-merTALEs tolerate multiple mutations in their target sequences. As shown inFIG. 10B, using the approach described in FIG. 2, the targetinglandscape of TALEs of 3 different sizes (18-mer, 14-mer and 10-mer) wasanalyzed. Shorter TALEs (14-mer and 10-mer) are progressively morespecific in their targeting but also reduced in activity by nearly anorder of magnitude. As shown in FIG. 10C and 10D, 10-mer TALEs show nearsingle-base mismatch resolution, losing almost all activity againsttargets bearing 2 mismatches (in the heat plot the target sequencepositions are labeled from 1-10 starting from the 5′ end). Takentogether, these data imply that engineering shorter TALEs can yieldhigher specificity in genome engineering applications, while therequirement for FokI dimerization in TALE nuclease applications isessential to avoid off-target effect. See Kim et al., Proceedings of theNational Academy of Sciences of the United States of America 93,1156-1160 (1996) and Pattanayak et al., Nature Methods 8, 765-770 (2011)each of which are hereby incorporated by reference in its entirety.

FIG. 8A-C is directed to high level specificity analysis processing flowfor calculation of normalized expression levels illustrated withexamples from experimental data. As shown in FIG. 8A, constructlibraries are generated with a biased distribution of binding sitesequences and random sequence 24 bp tags that will be incorporated intoreporter gene transcripts (top). The transcribed tags are highlydegenerate so that they should map many-to-one to Cas9 or TALE bindingsequences. The construct libraries are sequenced (3^(rd) level, left) toestablish which tags co-occur with binding sites, resulting in anassociation table of binding sites vs. transcribed tags (4^(th) level,left). Multiple construct libraries built for different binding sitesmay be sequenced at once using library barcodes (indicated here by thelight blue and light yellow colors; levels 1-4, left). A constructlibrary is then transfected into a cell population and a set ofdifferent Cas9/gRNA or TALE transcription factors are induced in samplesof the populations (2^(nd) level, right). One sample is always inducedwith a fixed TALE activator targeted to a fixed binding site sequencewithin the construct (top level, green box); this sample serves as apositive control (green sample, also indicated by a + sign). cDNAsgenerated from the reporter mRNA molecules in the induced samples arethen sequenced and analyzed to obtain tag counts for each tag in asample (3^(rd) and 4^(th) level, right). As with the construct librarysequencing, multiple samples, including the positive control, aresequenced and analyzed together by appending sample barcodes. Here thelight red color indicates one non-control sample that has been sequencedand analyzed with the positive control (green). Because only thetranscribed tags and not the construct binding sites appear in eachread, the binding site vs. tag association table obtained from constructlibrary sequencing is then used to tally up total counts of tagsexpressed from each binding site in each sample (5^(th) level). Thetallies for each non-positive control sample are then converted tonormalized expression levels for each binding site by dividing them bythe tallies obtained in the positive control sample. Examples of plotsof normalized expression levels by numbers of mismatches are provided inFIGS. 2B and 2E, and in FIG. 9A and FIG. 10B. Not covered in thisoverall process flow are several levels of filtering for erroneous tags,for tags not associable with a construct library, and for tagsapparently shared with multiple binding sites. FIG. 8B depicts exampledistributions of percentages of binding sites by numbers of mismatchesgenerated within a biased construct library. Left: Theoreticaldistribution. Right: Distribution observed from an actual TALE constructlibrary. FIG. 8C depicts example distributions of percentages of tagcounts aggregated to binding sites by numbers of mismatches. Left:Distribution observed from the positive control sample. Right:Distribution observed from a sample in which a non-control TALE wasinduced. As the positive control TALE binds to a fixed site in theconstruct, the distribution of aggregated tag counts closely reflectsthe distribution of binding sites in FIG. 8B, while the distribution isskewed to the left for the non-control TALE sample because sites withfewer mismatches induce higher expression levels. Below: Computing therelative enrichment between these by dividing the tag counts obtainedfor the target-TF by those obtained for the control-TF reveals theaverage expression level versus the number of mutations in the targetsite.

These results are further reaffirmed by specificity data generated usinga different Cas9-gRNA complex. As shown in FIG. 9A, a differentCas9-gRNA complex is tolerant to 1-3 mutations in its target sequence.As shown in FIG. 9B, the Cas9-gRNA complex is also largely insensitiveto point mutations, except those localized to the PAM sequence. As shownin FIG. 9C, introduction of 2 base mismatches however significantlyimpairs activity (in the heat plot the target sequence positions arelabeled from 1-23 starting from the 5′ end). As shown in FIG. 9D, it wasconfirmed using a nuclease mediated HR assay that the predicted PAM forthe S. pyogenes Cas9 is NGG and also NAG.

According to certain aspects, binding specificity is increased accordingto methods described herein. Because synergy between multiple complexesis a factor in target gene activation by Cas9N-VP64, transcriptionalregulation applications of Cas9N is naturally quite specific asindividual off-target binding events should have minimal effect.According to one aspect, off-set nicks are used in methods ofgenome-editing. A large majority of nicks seldom result in NHEJ events,(see Certo et al., Nature Methods 8, 671-676 (2011) hereby incorporatedby reference in its entirety) thus minimizing the effects of off-targetnicking. In contrast, inducing off-set nicks to generate double strandedbreaks (DSBs) is highly effective at inducing gene disruption. Accordingto certain aspects, 5′ overhangs generate more significant NHEJ eventsas opposed to 3′ overhangs. Similarly, 3′ overhangs favor HR over NHEJevents, although the total number of HR events is significantly lowerthan when a 5′ overhang is generated. Accordingly, methods are providedfor using nicks for homologous recombination and off-set nicks forgenerating double stranded breaks to minimize the effects of off-targetCas9-gRNA activity.

FIG. 3A-C is directed to multiplex off-set nicking and methods forreducing the off-target binding with the guide RNAs. As shown in FIG.3A, the traffic light reporter was used to simultaneously assay for HRand NHEJ events upon introduction of targeted nicks or breaks. DNAcleavage events resolved through the HDR pathway restore the GFPsequence, whereas mutagenic NHEJ causes frameshifts rendering the GFPout of frame and the downstream mCherry sequence in frame. For theassay, 14 gRNAs covering a 200 bp stretch of DNA: 7 targeting the sensestrand (U1-7) and 7 the antisense strand (D1-7) were designed. Using theCas9D10A mutant, which nicks the complementary strand, different two-waycombinations of the gRNAs were used to induce a range of programmed 5′or 3′ overhangs (the nicking sites for the 14 gRNAs are indicated). Asshown in FIG. 3B, inducing off-set nicks to generate double strandedbreaks (DSBs) is highly effective at inducing gene disruption. Notablyoff-set nicks leading to 5′ overhangs result in more NHEJ events asopposed to 3′ overhangs. As shown in FIG. 3C, generating 3′ overhangsalso favors the ratio of HR over NHEJ events, but the total number of HRevents is significantly lower than when a 5′ overhang is generated.

FIG. 11A-B is directed to Cas9D10A nickase mediated NHEJ. As shown inFIG. 11A, the traffic light reporter was used to assay NHEJ events uponintroduction of targeted nicks or double-stranded breaks. Briefly, uponintroduction of DNA cleavage events, if the break goes through mutagenicNHEJ, the GFP is translated out of frame and the downstream mCherrysequences are rendered in frame resulting in red fluorescence. 14 gRNAscovering a 200 bp stretch of DNA: 7 targeting the sense strand (U1-7)and 7 the antisense strand (D1-7) were designed. As shown in FIG. 11B,it was observed that unlike the wild-type Cas9 which results in DSBs androbust NHEJ across all targets, most nicks (using the Cas9D10A mutant)seldom result in NHEJ events. All 14 sites are located within acontiguous 200bp stretch of DNA and over 10-fold differences intargeting efficiencies were observed.

According to certain aspects, methods are described herein of modulatingexpression of a target nucleic acid in a cell that include introducingone or more, two or more or a plurality of foreign nucleic acids intothe cell. The foreign nucleic acids introduced into the cell encode fora guide RNA or guide RNAs, a nuclease-null Cas9 protein or proteins anda transcriptional regulator protein or domain. Together, a guide RNA, anuclease-null Cas9 protein and a transcriptional regulator protein ordomain are referred to as a co-localization complex as that term isunderstood by one of skill in the art to the extent that the guide RNA,the nuclease-null Cas9 protein and the transcriptional regulator proteinor domain bind to DNA and regulate expression of a target nucleic acid.According to certain additional aspects, the foreign nucleic acidsintroduced into the cell encode for a guide RNA or guide RNAs and a Cas9protein nickase. Together, a guide RNA and a Cas9 protein nickase arereferred to as a co-localization complex as that term is understood byone of skill in the art to the extent that the guide RNA and the Cas9protein nickase bind to DNA and nick a target nucleic acid.

Cells according to the present disclosure include any cell into whichforeign nucleic acids can be introduced and expressed as describedherein. It is to be understood that the basic concepts of the presentdisclosure described herein are not limited by cell type. Cellsaccording to the present disclosure include eukaryotic cells,prokaryotic cells, animal cells, plant cells, fungal cells, archaelcells, eubacterial cells and the like. Cells include eukaryotic cellssuch as yeast cells, plant cells, and animal cells. Particular cellsinclude mammalian cells. Further, cells include any in which it would bebeneficial or desirable to regulate a target nucleic acid. Such cellsmay include those which are deficient in expression of a particularprotein leading to a disease or detrimental condition. Such diseases ordetrimental conditions are readily known to those of skill in the art.According to the present disclosure, the nucleic acid responsible forexpressing the particular protein may be targeted by the methodsdescribed herein and a transcriptional activator resulting inupregulation of the target nucleic acid and corresponding expression ofthe particular protein. In this manner, the methods described hereinprovide therapeutic treatment.

Target nucleic acids include any nucleic acid sequence to which aco-localization complex as described herein can be useful to eitherregulate or nick. Target nucleic acids include genes. For purposes ofthe present disclosure, DNA, such as double stranded DNA, can includethe target nucleic acid and a co-localization complex can bind to orotherwise co-localize with the DNA at or adjacent or near the targetnucleic acid and in a manner in which the co-localization complex mayhave a desired effect on the target nucleic acid. Such target nucleicacids can include endogenous (or naturally occurring) nucleic acids andexogenous (or foreign) nucleic acids. One of skill based on the presentdisclosure will readily be able to identify or design guide RNAs andCas9 proteins which co-localize to a DNA including a target nucleicacid. One of skill will further be able to identify transcriptionalregulator proteins or domains which likewise co-localize to a DNAincluding a target nucleic acid. DNA includes genomic DNA, mitochondrialDNA, viral DNA or exogenous DNA.

Foreign nucleic acids (i.e. those which are not part of a cell's naturalnucleic acid composition) may be introduced into a cell using any methodknown to those skilled in the art for such introduction.

Such methods include transfection, transduction, viral transduction,microinjection, lipofection, nucleofection, nanoparticle bombardment,transformation, conjugation and the like. One of skill in the art willreadily understand and adapt such methods using readily identifiableliterature sources.

Transcriptional regulator proteins or domains which are transcriptionalactivators include VP16 and VP64 and others readily identifiable bythose skilled in the art based on the present disclosure.

Diseases and detrimental conditions are those characterized by abnormalloss of expression of a particular protein. Such diseases or detrimentalconditions can be treated by upregulation of the particular protein.Accordingly, methods of treating a disease or detrimental condition areprovided where the co-localization complex as described hereinassociates or otherwise binds to DNA including a target nucleic acid,and the transcriptional activator of the co-localization complexupregulates expression of the target nucleic acid. For exampleupregulating PRDM16 and other genes promoting brown fat differentiationand increased metabolic uptake can be used to treat metabolic syndromeor obesity. Activating anti-inflammatory genes are useful inautoimmunity and cardiovascular disease. Activating tumor suppressorgenes is useful in treating cancer. One of skill in the art will readilyidentify such diseases and detrimental conditions based on the presentdisclosure.

The following examples are set forth as being representative of thepresent disclosure. These examples are not to be construed as limitingthe scope of the present disclosure as these and other equivalentembodiments will be apparent in view of the present disclosure, figuresand accompanying claims.

EXAMPLE I Cas9 Mutants

Sequences homologous to Cas9 with known structure were searched toidentify candidate mutations in Cas9 that could ablate the naturalactivity of its RuvC and HNH domains. Using HHpred (world wide websitetoolkit.tuebingen.mpg.de/hhpred), the full sequence of Cas9 was queriedagainst the full Protein Data Bank (January 2013). This search returnedtwo different HNH endonucleases that had significant sequence homologyto the HNH domain of Cas9; PacI and a putative endonuclease (PDB IDs:3M7K and 4H9D respectively). These proteins were examined to findresidues involved in magnesium ion coordination. The correspondingresidues were then identified in the sequence alignment to Cas9. TwoMg-coordinating side-chains in each structure were identified thataligned to the same amino acid type in Cas9. They are 3M7K D92 and N113,and 4H9D D53 and N77. These residues corresponded to Cas9 D839 and N863.It was also reported that mutations of PacI residues D92 and N113 toalanine rendered the nuclease catalytically deficient. The Cas9mutations D839A and N863A were made based on this analysis.Additionally, HHpred also predicts homology between Cas9 and theN-terminus of a Thermus thermophilus RuvC (PDB ID: 4EP4). This sequencealignment covers the previously reported mutation D10A which eliminatesfunction of the RuvC domain in Cas9. To confirm this as an appropriatemutation, the metal binding residues were determined as before. In 4EP4,D7 helps to coordinate a magnesium ion. This position has sequencehomology corresponding to Cas9 D10, confirming that this mutation helpsremove metal binding, and thus catalytic activity from the Cas9 RuvCdomain

EXAMPLE II Plasmid Construction

The Cas9 mutants were generated using the Quikchange kit (Agilenttechnologies). The target gRNA expression constructs were either (1)directly ordered as individual gBlocks from IDT and cloned into thepCR-BluntII-TOPO vector (Invitrogen); or (2) custom synthesized byGenewiz; or (3) assembled using Gibson assembly of oligonucleotides intothe gRNA cloning vector (plasmid #41824). The vectors for the HRreporter assay involving a broken GFP were constructed by fusion PCRassembly of the GFP sequence bearing the stop codon and appropriatefragment assembled into the EGIP lentivector from Addgene (plasmid#26777). These lentivectors were then used to establish the GFP reporterstable lines. TALENs used in this study were constructed using standardprotocols. See Sanjana et al., Nature Protocols 7, 171-192 (2012) herebyincorporated by reference in its entirety. Cas9N and MS2 VP64 fusionswere performed using standard PCR fusion protocol procedures. Thepromoter luciferase constructs for OCT4 and REX1 were obtained fromAddgene (plasmid #17221 and plasmid #17222).

EXAMPLE III Cell Culture and Transfections

HEK 293T cells were cultured in Dulbecco' s modified Eagle's medium(DMEM, Invitrogen) high glucose supplemented with 10% fetal bovine serum(FBS, Invitrogen), penicillin/streptomycin (pen/strep, Invitrogen), andnon-essential amino acids (NEAA, Invitrogen). Cells were maintained at37° C. and 5% CO₂ in a humidified incubator.

Transfections involving nuclease assays were as follows: 0.4×10⁶ cellswere transfected with 2 μg Cas9 plasmid, 2 μg gRNA and/or 2 μg DNA donorplasmid using Lipofectamine 2000 as per the manufacturer's protocols.Cells were harvested 3 days after transfection and either analyzed byFACS, or for direct assay of genomic cuts the genomic DNA of ˜1×10⁶cells was extracted using DNAeasy kit (Qiagen). For these PCR wasconducted to amplify the targeting region with genomic DNA derived fromthe cells and amplicons were deep sequenced by MiSeq Personal Sequencer(Illumina) with coverage >200,000 reads. The sequencing data wasanalyzed to estimate NHEJ efficiencies.

For transfections involving transcriptional activation assays: 0.4×10⁶cells were transfected with (1) 2 μg Cas9N-VP64 plasmid, 2 μg gRNAand/or 0.25 μg of reporter construct; or (2) 2 μg Cas9N plasmid, 2 μgMS2-VP64, 2 μg gRNA-2XMS2aptamer and/or 0.25 μg of reporter construct.Cells were harvested 24-48 hrs post transfection and assayed using FACSor immunofluorescence methods, or their total RNA was extracted andthese were subsequently analyzed by RT-PCR. Here standard taqman probesfrom Invitrogen for OCT4 and REX1 were used, with normalization for eachsample performed against GAPDH.

For transfections involving transcriptional activation assays forspecificity profile of Cas9-gRNA complexes and TALEs: 0.4×10⁶ cells weretransfected with (1) 2 μg Cas9N-VP64 plasmid, 2 μg gRNA and 0.25 μg ofreporter library; or (2) 2 μg TALE-TF plasmid and 0.25 μg of reporterlibrary; or (3) 2 μg control-TF plasmid and 0.25 μg of reporter library.Cells were harvested 24 hrs post transfection (to avoid the stimulationof reporters being in saturation mode). Total RNA extraction wasperformed using RNAeasy-plus kit (Qiagen), and standard RT-per performedusing Superscript-III (Invitrogen). Libraries for next-generationsequencing were generated by targeted per amplification of thetranscript-tags.

EXAMPLE IV Computational and Sequence Analysis for Calculation ofCas9-TF and TALE-TF Reporter Expression Levels

The high-level logic flow for this process is depicted in FIG. 8A, andadditional details are given here. For details on construct librarycomposition, see FIGS. 8A (level 1) and 8B.

Sequencing: For Cas9 experiments, construct library (FIG. 8A, level 3,left) and reporter gene cDNA sequences (FIG. 8A, level 3, right) wereobtained as 150 bp overlapping paired end reads on an Illumina MiSeq,while for TALE experiments, corresponding sequences were obtained as 51bp non-overlapping paired end reads on an Illumina HiSeq.

Construct library sequence processing: Alignment: For Cas9 experiments,novoalign V2.07.17 (world wide website novocraft.com/main/index/php) wasused to align paired reads to a set of 250 bp reference sequences thatcorresponded to 234 bp of the constructs flanked by the pairs of 8 bplibrary barcodes (see FIG. 8A, 3^(rd) level, left). In the referencesequences supplied to novoalign, the 23 bp degenerate Cas9 binding siteregions and the 24 bp degenerate transcript tag regions (see FIG. 8A,first level) were specified as Ns, while the construct library barcodeswere explicitly provided. For TALE experiments, the same procedures wereused except that the reference sequences were 203 bp in length and thedegenerate binding site regions were 18 bp vs. 23 bp in length. Validitychecking: Novoalign output for comprised files in which left and rightreads for each read pair were individually aligned to the referencesequences. Only read pairs that were both uniquely aligned to thereference sequence were subjected to additional validity conditions, andonly read pairs that passed all of these conditions were retained. Thevalidity conditions included: (i) Each of the two construct librarybarcodes must align in at least 4 positions to a reference sequencebarcode, and the two barcodes must to the barcode pair for the sameconstruct library. (ii) All bases aligning to the N regions of thereference sequence must be called by novoalign as As, Cs, Gs or Ts. Notethat for neither Cas9 nor TALE experiments did left and right readsoverlap in a reference N region, so that the possibility of ambiguousnovoalign calls of these N bases did not arise. (iii) Likewise, nonovoalign-called inserts or deletions must appear in these regions. (iv)No Ts must appear in the transcript tag region (as these randomsequences were generated from As, Cs, and Gs only). Read pairs for whichany one of these conditions were violated were collected in a rejectedread pair file. These validity checks were implemented using custom perlscripts.

Induced sample reporter gene cDNA sequence processing: Alignment:SeqPrep (downloaded from world wide website github.com/jstjohn/SeqPrep)was first used to merge the overlapping read pairs to the 79 bp commonsegment, after which novoalign (version above) was used to align these79 bp common segments as unpaired single reads to a set of referencesequences (see FIG. 8A, 3^(rd) level, right) in which (as for theconstruct library sequencing) the 24 bp degenerate transcript tag wasspecified as Ns while the sample barcodes were explicitly provided. BothTALE and Cas9 cDNA sequence regions corresponded to the same 63 bpregions of cDNA flanked by pairs of 8 bp sample barcode sequences.Validity checking: The same conditions were applied as for constructlibrary sequencing (see above) except that: (a) Here, due prior SeqPrepmerging of read pairs, validity processing did not have to filter forunique alignments of both reads in a read pair but only for uniquealignments of the merged reads. (b) Only transcript tags appeared in thecDNA sequence reads, so that validity processing only applied these tagregions of the reference sequences and not also to a separate bindingsite region.

Assembly of table of binding sites vs. transcript tag associations:Custom perl was used to generate these tables from the validatedconstruct library sequences (FIG. 8A, 4^(th) level, left). Although the24bp tag sequences composed of A, C, and G bases should be essentiallyunique across a construct library (probability of sharing =˜2.8e−11),early analysis of binding site vs. tag associations revealed that anon-negligible fraction of tag sequences were in fact shared by multiplebinding sequences, likely mainly caused by a combination of sequenceerrors in the binding sequences, or oligo synthesis errors in the oligosused to generate the construct libraries. In addition to tag sharing,tags found associated with binding sites in validated read pairs mightalso be found in the construct library read pair reject file if it wasnot clear, due to barcode mismatches, which construct library they mightbe from. Finally, the tag sequences themselves might contain sequenceerrors. To deal with these sources of error, tags were categorized withthree attributes: (i) safe vs. unsafe, where unsafe meant the tag couldbe found in the construct library rejected read pair file; shared vs.nonshared, where shared meant the tag was found associated with multiplebinding site sequences, and 2+ vs. 1-only, where 2+ meant that the tagappeared at least twice among the validated construct library sequencesand so presumed to be less likely to contain sequence errors. Combiningthese three criteria yielded 8 classes of tags associated with eachbinding site, the most secure (but least abundant) class comprising onlysafe, nonshared, 2+ tags; and the least secure (but most abundant) classcomprising all tags regardless of safety, sharing, or number ofoccurrences.

Computation of normalized expression levels: Custom perl code was usedto implement the steps indicated in FIG. 8A, levels 5-6. First, tagcounts obtained for each induced sample were aggregated for each bindingsite, using the binding site vs. transcript tag table previouslycomputed for the construct library (see FIG. 8C). For each sample, theaggregated tag counts for each binding site were then divided by theaggregated tag counts for the positive control sample to generatenormalized expression levels. Additional considerations relevant tothese calculations included:

1. For each sample, a subset of “novel” tags were found among thevalidity-checked cDNA gene sequences that could not be found in thebinding site vs. transcript tag association table. These tags wereignored in the subsequent calculations.

2. The aggregations of tag counts described above were performed foreach of the eight classes of tags described above in binding site vs.transcript tag association table. Because the binding sites in theconstruct libraries were biased to generate sequences similar to acentral sequence frequently, but sequences with increasing numbers ofmismatches increasingly rarely, binding sites with few mismatchesgenerally aggregated to large numbers of tags, while binding sites withmore mismatches aggregated to smaller numbers. Thus, although use of themost secure tag class was generally desirable, evaluation of bindingsites with two or more mismatches might be based on small numbers oftags per binding site, making the secure counts and ratios lessstatistically reliable even if the tags themselves were more reliable.In such cases, all tags were used. Some compensation for thisconsideration obtains from the fact that the number of separateaggregated tag counts for n mismatching positions grew with the numberof combinations of mismatching positions (equal to

$\left. {\begin{pmatrix}L \\n\end{pmatrix}3^{n}} \right),$

and so dramatically increases with n; thus the averages of aggregatedtag counts for different numbers n of mismatches (shown in FIGS. 2b, 2e, and in FIGS. 9A and 10B) are based on a statistically very large setof aggregated tag counts for n≥2.

3. Finally, the binding site built into the TALE construct libraries was18 bp and tag associations were assigned based on these 18 bp sequences,but some experiments were conducted with TALEs programmed to bindcentral 14 bp or 10 bp regions within the 18 bp construct binding siteregions. In computing expression levels for these TALEs, tags wereaggregated to binding sites based on the corresponding regions of the 18bp binding sites in the association table, so that binding sitemismatches outside of this region were ignored.

EXAMPLE V RNA-guided SOX2 and NANOG Regulation Using Cas9_(N−) VP64

The sgRNA (aptamer-modified single guide RNA) tethering approachdescribed herein allows different effector domains to be recruited bydistinct sgRNAs so long as each sgRNA uses a different RNA-proteininteraction pair, enabling multiplex gene regulation using the sameCas9N-protein. For the FIG. 12A SOX2 and FIG. 12B NANOG genes, 10 gRNAswere designed targeting a ˜1 kb stretch of DNA upstream of thetranscription start site. The DNase hypersensitive sites are highlightedin green. Transcriptional activation via qPCR of the endogenous geneswas assayed. In both instances, while introduction of individual gRNAsmodestly stimulated transcription, multiple gRNAs acted synergisticallyto stimulate robust multi-fold transcriptional activation. Data aremeans +/− SEM (N=3). As shown in FIGS. 12A-B, two additional genes, SOX2and NANOG, were regulated via sgRNAs targeting within an upstream ˜1 kbstretch of promoter DNA. The sgRNAs proximal to the transcriptionalstart site resulted in robust gene activation.

EXAMPLE VI Evaluating the Landscape of targeting by Cas9-gRNA Complexes

Using the approach described in FIG. 2, the targeting landscape of twoadditional Cas9-gRNA complexes (FIG. 13A-C) and (FIG. 13D-F) wasanalyzed. The two gRNAs have vastly different specificity profiles withgRNA2 tolerating up to 2-3 mismatches and gRNA3 only up to 1. Theseaspects are reflected in both the one base mismatch (FIG. 13B, 13E) andtwo base mismatch plots (FIG. 13C, 13F). In FIGS. 13C and 13F, basemismatch pairs for which insufficient data were available to calculate anormalized expression level are indicated as gray boxes containing an‘x’, while, to improve data display, mismatch pairs whose normalizedexpression levels are outliers that exceed the top of the color scaleare indicated as yellow boxes containing an asterisk ‘*’. Statisticalsignificance symbols are: *** for P<0.0005/n, ** for P<0.005/n, * forP<0.05/n, and N.S. (Non-Significant) for P>=.05/n, where n is the numberof comparisons (refer Table 2).

EXAMPLE VII Validations, Specificity of Reporter Assay

As shown in FIGS. 14A-C, specificity data was generated using twodifferent sgRNA:Cas9 complexes. It was confirmed that the assay wasspecific for the sgRNA being evaluated, as a corresponding mutant sgRNAwas unable to stimulate the reporter library. FIG. 14A: The specificityprofile of two gRNAs (wild-type and mutant; sequence differences arehighlighted in red) were evaluated using a reporter library designedagainst the wild-type gRNA target sequence. FIG. 14B: It was confirmedthat this assay was specific for the gRNA being evaluated (datare-plotted from FIG. 13D), as the corresponding mutant gRNA is unable tostimulate the reporter library. Statistical significance symbols are:*** for P<0.0005/n, ** for P<0.005/n, * for P<0.05/n, and N.S.(Non-Significant) for P>=.05/n, where n is the number of comparisons(refer Table 2). Different sgRNAs can have different specificityprofiles (FIGS. 13A, 13D), specifically, sgRNA2 tolerates up to 3mismatches and sgRNA3 only up to 1. The greatest sensitivity tomismatches was localized to the 3′ end of the spacer, albeit mismatchesat other positions were also observed to affect activity.

EXAMPLE VIII Validations, Single and Double-base gRNA Mismatches

As shown in FIGS. 15A, 15B-1, 15B-2, 15C, 15D-1, and 15D-2, it wasconfirmed by targeted experiments that single-base mismatches within 12bp of the 3′ end of the spacer in the assayed sgRNAs resulted indetectable targeting. However, 2 bp mismatches in this region resultedin significant loss of activity. Using a nuclease assay, 2 independentgRNAs were tested: gRNA2 (FIGS. 15A-15B-2) and gRNA3 (FIGS. 15C-15D-2)bearing single or double-base mismatches (highlighted in red) in thespacer sequence versus the target. It was confirmed that single-basemismatches within 12 bp of the 3′ end of the spacer in the assayed gRNAsresult in detectable targeting, however 2 bp mismatches in this regionresult in rapid loss of activity. These results further highlight thedifferences in specificity profiles between different gRNAs consistentwith the results in FIG. 13. Data are means +/− SEM (N=3).

EXAMPLE IX Validations, 5′ gRNA Truncations

As shown in FIGS. 16A, 16B-1, 16B2, 16C, 16D-1, and 16D-2, truncationsin the 5′ portion of the spacer resulted in retention of sgRNA activity.Using a nuclease assay, 2 independent gRNA were tested: gRNA1 (FIGS.16A-16B-2) and gRNA3 (FIGS. 16C-16D-2) bearing truncations at the 5′ endof their spacer. It was observed that 1-3 bp 5′ truncations are welltolerated, but larger deletions lead to loss of activity. Data are means+/− SEM (N=3).

EXAMPLE X Validations, S. pyogenes PAM

As shown in FIGS. 17A-B, it was confirmed using a nuclease mediated HRassay that the PAM for the S. pyogenes Cas9 is NGG and also NAG. Dataare means +/− SEM (N=3). According to an additional investigation, agenerated set of about 190K Cas9 targets in human exons that had noalternate NGG targets sharing the last 13 nt of the targeting sequencewas scanned for the presence of alternate NAG sites or for NGG siteswith a mismatch in the prior 13 nt. Only 0.4% were found to have no suchalternate targets.

EXAMPLE XI Validations, TALE Mutations

Using a nuclease mediated HR assay (FIGS. 18A-B) it was confirmed that18-mer TALEs tolerate multiple mutations in their target sequences. Asshown in FIGS. 18A-B certain mutations in the middle of the target leadto higher TALE activity, as determined via targeted experiments in anuclease assay.

EXAMPLE XII TALE Monomer Specificity Versus TALE Protein Specificity

To decouple the role of individual repeat-variable diresidues (RVDs), itwas confirmed that choice of RVDs did contribute to base specificity butTALE specificity is also a function of the binding energy of the proteinas a whole. FIGS. 19A-19C-2 show a comparison of TALE monomerspecificity versus TALE protein specificity. FIG. 19A: Using amodification of approach described in FIG. 2, the targeting landscape of2 14-mer TALE-TFs bearing a contiguous set of 6 NI or 6 NH repeats wasanalyzed. In this approach, a reduced library of reporters bearing adegenerate 6-mer sequence in the middle was created and used to assaythe TALE-TF specificity. FIGS. 19B-1-19C-2: In both instances, it wasnoted that the expected target sequence is enriched (i.e. one bearing 6As for NI repeats, and 6 Gs for NH repeats). Each of these TALEs stilltolerate 1-2 mismatches in the central 6-mer target sequence. Whilechoice of monomers does contribute to base specificity, TALE specificityis also a function of the binding energy of the protein as a whole.According to one aspect, shorter engineered TALEs or TALEs bearing acomposition of high and low affinity monomers result in higherspecificity in genome engineering applications and FokI dimerization innuclease applications allows for further reduction in off-target effectswhen using shorter TALEs.

EXAMPLE XIII Off-set Nicking, Native Locus

FIGS. 20A-B shows data related to off-set nicking. In the context ofgenome-editing, off-set nicks were created to generate DSBs. A largemajority of nicks do not result in non-homologous end joining (NHEJ)mediated indels and thus when inducing off-set nicks, off-target singlenick events will likely result in very low indel rates. Inducing off-setnicks to generate DSBs is effective at inducing gene disruption at bothintegrated reporter loci and at the native AAVS1 genomic locus.

FIG. 20A: The native AAVS1 locus with 8 gRNAs covering a 200 bp stretchof DNA was targeted: 4 targeting the sense strand (s1-4) and 4 theantisense strand (as1-4). Using the Cas9D10A mutant, which nicks thecomplementary strand, different two-way combinations of the gRNAs wasused to induce a range of programmed 5′ or 3′ overhangs. FIG. 20B: Usinga Sanger sequencing based assay, it was observed that while single gRNAsdid not induce detectable NHEJ events, inducing off-set nicks togenerate DSBs is highly effective at inducing gene disruption. Notablyoff-set nicks leading to 5′ overhangs result in more NHEJ events asopposed to 3′ overhangs. The number of Sanger sequencing clones ishighlighted above the bars, and the predicted overhang lengths areindicated below the corresponding x-axis legends.

EXAMPLE XIV Off-set Nicking, NHEJ Profiles

FIG. 21A-C is directed to off-set nicking and NHEJ profiles.Representative Sanger sequencing results of three different off-setnicking combinations is shown with positions of the targeting gRNAshighlighted by boxes. Furthermore, consistent with the standard modelfor homologous recombination (HR) mediated repair, engineering of 5′overhangs via off-set nicks generated more robust NHEJ events than 3′overhangs (FIG. 3B). In addition to a stimulation of NHEJ, robustinduction of HR was observed when the 5′ overhangs were created.Generation of 3′ overhangs did not result in improvement of HR rates(FIG. 3C).

EXAMPLE XV

EXAMPLE XVI Table 2

Summary of Statistical Analysis of Cas9-gRNA and TALE Specificity Data

EXAMPLE XVII Sequences of Proteins and RNAs in the Examples

A. Sequences of the Cas9_(N−) VP64 activator constructs based on the m4mutant are displayed below. Three versions were constructed with theCas9_(m4) ^(VP64) and Cas9_(m4) ^(vP64)N fusion protein formats showinghighest activity. Corresponding vectors for the m3 and m2 mutants (FIG.4A) were also constructed (NLS and VP64 domains are highlighted).

>Cas9_(m4) ^(VP64) (SEQ ID NO: 2)gccaccATGGACAAGAAGTACTCCATTGGGCTCGCTATCGGCACAAACAGCGTCGGCTGGGCCGTCATTACGGACGAGTACAAGGTGCCGAGCAAAAAATTCAAAGTTCTGGGCAATACCGATCGCCACAGCATAAAGAAGAACCTCATTGGCGCCCTCCTGTTCGACTCCGGGGAGACGGCCGAAGCCACGCGGCTCAAAAGAACAGCACGGCGCAGATATACCCGCAGAAAGAATCGGATCTGCTACCTGCAGGAGATCTTTAGTAATGAGATGGCTAAGGTGGATGACTCTTTCTTCCATAGGCTGGAGGAGTCCTTTTTGGTGGAGGAGGATAAAAAGCACGAGCGCCACCCAATCTTTGGCAATATCGTGGACGAGGTGGCGTACCATGAAAAGTACCCAACCATATATCATCTGAGGAAGAAGCTTGTAGACAGTACTGATAAGGCTGACTTGCGGTTGATCTATCTCGCGCTGGCGCATATGATCAAATTTCGGGGACACTTCCTCATCGAGGGGGACCTGAACCCAGACAACAGCGATGTCGACAAACTCTTTATCCAACTGGTTCAGACTTACAATCAGCTTTTCGAAGAGAACCCGATCAACGCATCCGGAGTTGACGCCAAAGCAATCCTGAGCGCTAGGCTGTCCAAATCCCGGCGGCTCGAAAACCTCATCGCACAGCTCCCTGGGGAGAAGAAGAACGGCCTGTTTGGTAATCTTATCGCCCTGTCACTCGGGCTGACCCCCAACTTTAAATCTAACTTCGACCTGGCCGAAGATGCCAAGCTTCAACTGAGCAAAGACACCTACGATGATGATCTCGACAATCTGCTGGCCCAGATCGGCGACCAGTACGCAGACCTTTTTTTGGCGGCAAAGAACCTGTCAGACGCCATTCTGCTGAGTGATATTCTGCGAGTGAACACGGAGATCACCAAAGCTCCGCTGAGCGCTAGTATGATCAAGCGCTATGATGAGCACCACCAAGACTTGACTTTGCTGAAGGCCCTTGTCAGACAGCAACTGCCTGAGAAGTACAAGGAAATTTTCTTCGATCAGTCTAAAAATGGCTACGCCGGATACATTGACGGCGGAGCAAGCCAGGAGGAATTTTACAAATTTATTAAGCCCATCTTGGAAAAAATGGACGGCACCGAGGAGCTGCTGGTAAAGCTTAACAGAGAAGATCTGTTGCGCAAACAGCGCACTTTCGACAATGGAAGCATCCCCCACCAGATTCACCTGGGCGAACTGCACGCTATCCTCAGGCGGCAAGAGGATTTCTACCCCTTTTTGAAAGATAACAGGGAAAAGATTGAGAAAATCCTCACATTTCGGATACCCTACTATGTAGGCCCCCTCGCCCGGGGAAATTCCAGATTCGCGTGGATGACTCGCAAATCAGAAGAGACCATCACTCCCTGGAACTTCGAGGAAGTCGTGGATAAGGGGGCCTCTGCCCAGTCCTTCATCGAAAGGATGACTAACTTTGATAAAAATCTGCCTAACGAAAAGGTGCTTCCTAAACACTCTCTGCTGTACGAGTACTTCACAGTTTATAACGAGCTCACCAAGGTCAAATACGTCACAGAAGGGATGAGAAAGCCAGCATTCCTGTCTGGAGAGCAGAAGAAAGCTATCGTGGACCTCCTCTTCAAGACGAACCGGAAAGTTACCGTGAAACAGCTCAAAGAAGACTATTTCAAAAAGATTGAATGTTTCGACTCTGTTGAAATCAGCGGAGTGGAGGATCGCTTCAACGCATCCCTGGGAACGTATCACGATCTCCTGAAAATCATTAAAGACAAGGACTTCCTGGACAATGAGGAGAACGAGGACATTCTTGAGGACATTGTCCTCACCCTTACGTTGTTTGAAGATAGGGAGATGATTGAAGAACGCTTGAAAACTTACGCTCATCTCTTCGACGACAAAGTCATGAAACAGCTCAAGAGGCGCCGATATACAGGATGGGGGCGGCTGTCAAGAAAACTGATCAATGGGATCCGAGACAAGCAGAGTGGAAAGACAATCCTGGATTTTCTTAAGTCCGATGGATTTGCCAACCGGAACTTCATGCAGTTGATCCATGATGACTCTCTCACCTTTAAGGAGGACATCCAGAAAGCACAAGTTTCTGGCCAGGGGGACAGTCTTCACGAGCACATCGCTAATCTTGCAGGTAGCCCAGCTATCAAAAAGGGAATACTGCAGACCGTTAAGGTCGTGGATGAACTCGTCAAAGTAATGGGAAGGCATAAGCCCGAGAATATCGTTATCGAGATGGCCCGAGAGAACCAAACTACCCAGAAGGGACAGAAGAACAGTAGGGAAAGGATGAAGAGGATTGAAGAGGGTATAAAAGAACTGGGGTCCCAAATCCTTAAGGAACACCCAGTTGAAAACACCCAGCTTCAGAATGAGAAGCTCTACCTGTACTACCTGCAGAACGGCAGGGACATGTACGTGGATCAGGAACTGGACATCAATCGGCTCTCCGACTACGACGTGGCTGCTATCGTGCCCCAGTCTTTTCTCAAAGATGATTCTATTGATAATAAAGTGTTGACAAGATCCGATAAAgcTAGAGGGAAGAGTGATAACGTCCCCTCAGAAGAAGTTGTCAAGAAAATGAAAAATTATTGGCGGCAGCTGCTGAACGCCAAACTGATCACACAACGGAAGTTCGATAATCTGACTAAGGCTGAACGAGGTGGCCTGTCTGAGTTGGATAAAGCCGGCTTCATCAAAAGGCAGCTTGTTGAGACACGCCAGATCACCAAGCACGTGGCCCAAATTCTCGATTCACGCATGAACACCAAGTACGATGAAAATGACAAACTGATTCGAGAGGTGAAAGTTATTACTCTGAAGTCTAAGCTGGTCTCAGATTTCAGAAAGGACTTTCAGTTTTATAAGGTGAGAGAGATCAACAATTACCACCATGCGCATGATGCCTACCTGAATGCAGTGGTAGGCACTGCACTTATCAAAAAATATCCCAAGCTTGAATCTGAATTTGTTTACGGAGACTATAAAGTGTACGATGTTAGGAAAATGATCGCAAAGTCTGAGCAGGAAATAGGCAAGGCCACCGCTAAGTACTTCTTTTACAGCAATATTATGAATTTTTTCAAGACCGAGATTACACTGGCCAATGGAGAGATTCGGAAGCGACCACTTATCGAAACAAACGGAGAAACAGGAGAAATCGTGTGGGACAAGGGTAGGGATTTCGCGACAGTCCGGAAGGTCCTGTCCATGCCGCAGGTGAACATCGTTAAAAAGACCGAAGTACAGACCGGAGGCTTCTCCAAGGAAAGTATCCTCCCGAAAAGGAACAGCGACAAGCTGATCGCACGCAAAAAAGATTGGGACCCCAAGAAATACGGCGGATTCGATTCTCCTACAGTCGCTTACAGTGTACTGGTTGTGGCCAAAGTGGAGAAAGGGAAGTCTAAAAAACTCAAAAGCGTCAAGGAACTGCTGGGCATCACAATCATGGAGCGATCAAGCTTCGAAAAAAACCCCATCGACTTTCTCGAGGCGAAAGGATATAAAGAGGTCAAAAAAGACCTCATCATTAAGCTTCCCAAGTACTCTCTCTTTGAGCTTGAAAACGGCCGGAAACGAATGCTCGCTAGTGCGGGCGAGCTGCAGAAAGGTAACGAGCTGGCACTGCCCTCTAAATACGTTAATTTCTTGTATCTGGCCAGCCACTATGAAAAGCTCAAAGGGTCTCCCGAAGATAATGAGCAGAAGCAGCTGTTCGTGGAACAACACAAACACTACCTTGATGAGATCATCGAGCAAATAAGCGAATTCTCCAAAAGAGTGATCCTCGCCGACGCTAACCTCGATAAGGTGCTTTCTGCTTACAATAAGCACAGGGATAAGCCCATCAGGGAGCAGGCAGAAAACATTATCCACTTGTTTACTCTGACCAACTTGGGCGCGCCTGCAGCCTTCAAGTACTTCGACACCACCATAGACAGAAAGCGGTACACCTCTACAAAGGAGGTCCTGGACGCCACACTGATTCATCAGTCAATTACGGGGCTCTATGAAACAAGAATCGACCTCTCTCAGCTCGGTGGAGACAGCAGGGCTGACCCCAAGAAGAAGAGGAAGGTGGAGGCCAGCGGTTCCGGACGGGCTGACGCATTGGACGATTTTGATCTGGATATGCTGGGAAGTGACGCCCTCGATGATTTTGACCTTGACATGCTTGGTTCGGATGCCCTTGATGACTTTGACCTCGACATGCTCGGCAGTGACGCCCTTGATGATTTCGACCTGGACATGCTGATTAACTCTAGATGA >Cas9_(m4) ^(VP64)N Sequences(SEQ ID NO: 3) gccaccATGCCCAAGAAGAAGAGGAAGGTGGGAAGGGGGATGGACAAGAAGTACTCCATTGGGCTCGCTATCGGCACAAACAGCGTCGGCTGGGCCGTCATTACGGACGAGTACAAGGTGCCGAGCAAAAAATTCAAAGTTCTGGGCAATACCGATCGCCACAGCATAAAGAAGAACCTCATTGGCGCCCTCCTGTTCGACTCCGGGGAGACGGCCGAAGCCACGCGGCTCAAAAGAACAGCACGGCGCAGATATACCCGCAGAAAGAATCGGATCTGCTACCTGCAGGAGATCTTTAGTAATGAGATGGCTAAGGTGGATGACTCTTTCTTCCATAGGCTGGAGGAGTCCTTTTTGGTGGAGGAGGATAAAAAGCACGAGCGCCACCCAATCTTTGGCAATATCGTGGACGAGGTGGCGTACCATGAAAAGTACCCAACCATATATCATCTGAGGAAGAAGCTTGTAGACAGTACTGATAAGGCTGACTTGCGGTTGATCTATCTCGCGCTGGCGCATATGATCAAATTTCGGGGACACTTCCTCATCGAGGGGGACCTGAACCCAGACAACAGCGATGTCGACAAACTCTTTATCCAACTGGTTCAGACTTACAATCAGCTTTTCGAAGAGAACCCGATCAACGCATCCGGAGTTGACGCCAAAGCAATCCTGAGCGCTAGGCTGTCCAAATCCCGGCGGCTCGAAAACCTCATCGCACAGCTCCCTGGGGAGAAGAAGAACGGCCTGTTTGGTAATCTTATCGCCCTGTCACTCGGGCTGACCCCCAACTTTAAATCTAACTTCGACCTGGCCGAAGATGCCAAGCTTCAACTGAGCAAAGACACCTACGATGATGATCTCGACAATCTGCTGGCCCAGATCGGCGACCAGTACGCAGACCTTTTTTTGGCGGCAAAGAACCTGTCAGACGCCATTCTGCTGAGTGATATTCTGCGAGTGAACACGGAGATCACCAAAGCTCCGCTGAGCGCTAGTATGATCAAGCGCTATGATGAGCACCACCAAGACTTGACTTTGCTGAAGGCCCTTGTCAGACAGCAACTGCCTGAGAAGTACAAGGAAATTTTCTTCGATCAGTCTAAAAATGGCTACGCCGGATACATTGACGGCGGAGCAAGCCAGGAGGAATTTTACAAATTTATTAAGCCCATCTTGGAAAAAATGGACGGCACCGAGGAGCTGCTGGTAAAGCTTAACAGAGAAGATCTGTTGCGCAAACAGCGCACTTTCGACAATGGAAGCATCCCCCACCAGATTCACCTGGGCGAACTGCACGCTATCCTCAGGCGGCAAGAGGATTTCTACCCCTTTTTGAAAGATAACAGGGAAAAGATTGAGAAAATCCTCACATTTCGGATACCCTACTATGTAGGCCCCCTCGCCCGGGGAAATTCCAGATTCGCGTGGATGACTCGCAAATCAGAAGAGACCATCACTCCCTGGAACTTCGAGGAAGTCGTGGATAAGGGGGCCTCTGCCCAGTCCTTCATCGAAAGGATGACTAACTTTGATAAAAATCTGCCTAACGAAAAGGTGCTTCCTAAACACTCTCTGCTGTACGAGTACTTCACAGTTTATAACGAGCTCACCAAGGTCAAATACGTCACAGAAGGGATGAGAAAGCCAGCATTCCTGTCTGGAGAGCAGAAGAAAGCTATCGTGGACCTCCTCTTCAAGACGAACCGGAAAGTTACCGTGAAACAGCTCAAAGAAGACTATTTCAAAAAGATTGAATGTTTCGACTCTGTTGAAATCAGCGGAGTGGAGGATCGCTTCAACGCATCCCTGGGAACGTATCACGATCTCCTGAAAATCATTAAAGACAAGGACTTCCTGGACAATGAGGAGAACGAGGACATTCTTGAGGACATTGTCCTCACCCTTACGTTGTTTGAAGATAGGGAGATGATTGAAGAACGCTTGAAAACTTACGCTCATCTCTTCGACGACAAAGTCATGAAACAGCTCAAGAGGCGCCGATATACAGGATGGGGGCGGCTGTCAAGAAAACTGATCAATGGGATCCGAGACAAGCAGAGTGGAAAGACAATCCTGGATTTTCTTAAGTCCGATGGATTTGCCAACCGGAACTTCATGCAGTTGATCCATGATGACTCTCTCACCTTTAAGGAGGACATCCAGAAAGCACAAGTTTCTGGCCAGGGGGACAGTCTTCACGAGCACATCGCTAATCTTGCAGGTAGCCCAGCTATCAAAAAGGGAATACTGCAGACCGTTAAGGTCGTGGATGAACTCGTCAAAGTAATGGGAAGGCATAAGCCCGAGAATATCGTTATCGAGATGGCCCGAGAGAACCAAACTACCCAGAAGGGACAGAAGAACAGTAGGGAAAGGATGAAGAGGATTGAAGAGGGTATAAAAGAACTGGGGTCCCAAATCCTTAAGGAACACCCAGTTGAAAACACCCAGCTTCAGAATGAGAAGCTCTACCTGTACTACCTGCAGAACGGCAGGGACATGTACGTGGATCAGGAACTGGACATCAATCGGCTCTCCGACTACGACGTGGCTGCTATCGTGCCCCAGTCTTTTCTCAAAGATGATTCTATTGATAATAAAGTGTTGACAAGATCCGATAAAgcTAGAGGGAAGAGTGATAACGTCCCCTCAGAAGAAGTTGTCAAGAAAATGAAAAATTATTGGCGGCAGCTGCTGAACGCCAAACTGATCACACAACGGAAGTTCGATAATCTGACTAAGGCTGAACGAGGTGGCCTGTCTGAGTTGGATAAAGCCGGCTTCATCAAAAGGCAGCTTGTTGAGACACGCCAGATCACCAAGCACGTGGCCCAAATTCTCGATTCACGCATGAACACCAAGTACGATGAAAATGACAAACTGATTCGAGAGGTGAAAGTTATTACTCTGAAGTCTAAGCTGGTCTCAGATTTCAGAAAGGACTTTCAGTTTTATAAGGTGAGAGAGATCAACAATTACCACCATGCGCATGATGCCTACCTGAATGCAGTGGTAGGCACTGCACTTATCAAAAAATATCCCAAGCTTGAATCTGAATTTGTTTACGGAGACTATAAAGTGTACGATGTTAGGAAAATGATCGCAAAGTCTGAGCAGGAAATAGGCAAGGCCACCGCTAAGTACTTCTTTTACAGCAATATTATGAATTTTTTCAAGACCGAGATTACACTGGCCAATGGAGAGATTCGGAAGCGACCACTTATCGAAACAAACGGAGAAACAGGAGAAATCGTGTGGGACAAGGGTAGGGATTTCGCGACAGTCCGGAAGGTCCTGTCCATGCCGCAGGTGAACATCGTTAAAAAGACCGAAGTACAGACCGGAGGCTTCTCCAAGGAAAGTATCCTCCCGAAAAGGAACAGCGACAAGCTGATCGCACGCAAAAAAGATTGGGACCCCAAGAAATACGGCGGATTCGATTCTCCTACAGTCGCTTACAGTGTACTGGTTGTGGCCAAAGTGGAGAAAGGGAAGTCTAAAAAACTCAAAAGCGTCAAGGAACTGCTGGGCATCACAATCATGGAGCGATCAAGCTTCGAAAAAAACCCCATCGACTTTCTCGAGGCGAAAGGATATAAAGAGGTCAAAAAAGACCTCATCATTAAGCTTCCCAAGTACTCTCTCTTTGAGCTTGAAAACGGCCGGAAACGAATGCTCGCTAGTGCGGGCGAGCTGCAGAAAGGTAACGAGCTGGCACTGCCCTCTAAATACGTTAATTTCTTGTATCTGGCCAGCCACTATGAAAAGCTCAAAGGGTCTCCCGAAGATAATGAGCAGAAGCAGCTGTTCGTGGAACAACACAAACACTACCTTGATGAGATCATCGAGCAAATAAGCGAATTCTCCAAAAGAGTGATCCTCGCCGACGCTAACCTCGATAAGGTGCTTTCTGCTTACAATAAGCACAGGGATAAGCCCATCAGGGAGCAGGCAGAAAACATTATCCACTTGTTTACTCTGACCAACTTGGGCGCGCCTGCAGCCTTCAAGTACTTCGACACCACCATAGACAGAAAGCGGTACACCTCTACAAAGGAGGTCCTGGACGCCACACTGATTCATCAGTCAATTACGGGGCTCTATGAAACAAGAATCGACCTCTCTCAGCTCGGTGGAGACAGCAGGGCTGACCCCAAGAAGAAGAGGAAGGTGGAGGCCAGCGGTTCCGGACGGGCTGACGCATTGGACGATTTTGATCTGGATATGCTGGGAAGTGACGCCCTCGATGATTTTGACCTTGACATGCTTGGTTCGGATGCCCTTGATGACTTTGACCTCGACATGCTCGGCAGTGACGCCCTTGATGATTTCGACCTGGACATGCTG ATTAACTCTAGATGA>Cas9_(m4) ^(VP64)C (SEQ ID NO: 4)gccaccATGGACAAGAAGTACTCCATTGGGCTCGCTATCGGCACAAACAGCGTCGGCTGGGCCGTCATTACGGACGAGTACAAGGTGCCGAGCAAAAAATTCAAAGTTCTGGGCAATACCGATCGCCACAGCATAAAGAAGAACCTCATTGGCGCCCTCCTGTTCGACTCCGGGGAGACGGCCGAAGCCACGCGGCTCAAAAGAACAGCACGGCGCAGATATACCCGCAGAAAGAATCGGATCTGCTACCTGCAGGAGATCTTTAGTAATGAGATGGCTAAGGTGGATGACTCTTTCTTCCATAGGCTGGAGGAGTCCTTTTTGGTGGAGGAGGATAAAAAGCACGAGCGCCACCCAATCTTTGGCAATATCGTGGACGAGGTGGCGTACCATGAAAAGTACCCAACCATATATCATCTGAGGAAGAAGCTTGTAGACAGTACTGATAAGGCTGACTTGCGGTTGATCTATCTCGCGCTGGCGCATATGATCAAATTTCGGGGACACTTCCTCATCGAGGGGGACCTGAACCCAGACAACAGCGATGTCGACAAACTCTTTATCCAACTGGTTCAGACTTACAATCAGCTTTTCGAAGAGAACCCGATCAACGCATCCGGAGTTGACGCCAAAGCAATCCTGAGCGCTAGGCTGTCCAAATCCCGGCGGCTCGAAAACCTCATCGCACAGCTCCCTGGGGAGAAGAAGAACGGCCTGTTTGGTAATCTTATCGCCCTGTCACTCGGGCTGACCCCCAACTTTAAATCTAACTTCGACCTGGCCGAAGATGCCAAGCTTCAACTGAGCAAAGACACCTACGATGATGATCTCGACAATCTGCTGGCCCAGATCGGCGACCAGTACGCAGACCTTTTTTTGGCGGCAAAGAACCTGTCAGACGCCATTCTGCTGAGTGATATTCTGCGAGTGAACACGGAGATCACCAAAGCTCCGCTGAGCGCTAGTATGATCAAGCGCTATGATGAGCACCACCAAGACTTGACTTTGCTGAAGGCCCTTGTCAGACAGCAACTGCCTGAGAAGTACAAGGAAATTTTCTTCGATCAGTCTAAAAATGGCTACGCCGGATACATTGACGGCGGAGCAAGCCAGGAGGAATTTTACAAATTTATTAAGCCCATCTTGGAAAAAATGGACGGCACCGAGGAGCTGCTGGTAAAGCTTAACAGAGAAGATCTGTTGCGCAAACAGCGCACTTTCGACAATGGAAGCATCCCCCACCAGATTCACCTGGGCGAACTGCACGCTATCCTCAGGCGGCAAGAGGATTTCTACCCCTTTTTGAAAGATAACAGGGAAAAGATTGAGAAAATCCTCACATTTCGGATACCCTACTATGTAGGCCCCCTCGCCCGGGGAAATTCCAGATTCGCGTGGATGACTCGCAAATCAGAAGAGACCATCACTCCCTGGAACTTCGAGGAAGTCGTGGATAAGGGGGCCTCTGCCCAGTCCTTCATCGAAAGGATGACTAACTTTGATAAAAATCTGCCTAACGAAAAGGTGCTTCCTAAACACTCTCTGCTGTACGAGTACTTCACAGTTTATAACGAGCTCACCAAGGTCAAATACGTCACAGAAGGGATGAGAAAGCCAGCATTCCTGTCTGGAGAGCAGAAGAAAGCTATCGTGGACCTCCTCTTCAAGACGAACCGGAAAGTTACCGTGAAACAGCTCAAAGAAGACTATTTCAAAAAGATTGAATGTTTCGACTCTGTTGAAATCAGCGGAGTGGAGGATCGCTTCAACGCATCCCTGGGAACGTATCACGATCTCCTGAAAATCATTAAAGACAAGGACTTCCTGGACAATGAGGAGAACGAGGACATTCTTGAGGACATTGTCCTCACCCTTACGTTGTTTGAAGATAGGGAGATGATTGAAGAACGCTTGAAAACTTACGCTCATCTCTTCGACGACAAAGTCATGAAACAGCTCAAGAGGCGCCGATATACAGGATGGGGGCGGCTGTCAAGAAAACTGATCAATGGGATCCGAGACAAGCAGAGTGGAAAGACAATCCTGGATTTTCTTAAGTCCGATGGATTTGCCAACCGGAACTTCATGCAGTTGATCCATGATGACTCTCTCACCTTTAAGGAGGACATCCAGAAAGCACAAGTTTCTGGCCAGGGGGACAGTCTTCACGAGCACATCGCTAATCTTGCAGGTAGCCCAGCTATCAAAAAGGGAATACTGCAGACCGTTAAGGTCGTGGATGAACTCGTCAAAGTAATGGGAAGGCATAAGCCCGAGAATATCGTTATCGAGATGGCCCGAGAGAACCAAACTACCCAGAAGGGACAGAAGAACAGTAGGGAAAGGATGAAGAGGATTGAAGAGGGTATAAAAGAACTGGGGTCCCAAATCCTTAAGGAACACCCAGTTGAAAACACCCAGCTTCAGAATGAGAAGCTCTACCTGTACTACCTGCAGAACGGCAGGGACATGTACGTGGATCAGGAACTGGACATCAATCGGCTCTCCGACTACGACGTGGCTGCTATCGTGCCCCAGTCTTTTCTCAAAGATGATTCTATTGATAATAAAGTGTTGACAAGATCCGATAAAgcTAGAGGGAAGAGTGATAACGTCCCCTCAGAAGAAGTTGTCAAGAAAATGAAAAATTATTGGCGGCAGCTGCTGAACGCCAAACTGATCACACAACGGAAGTTCGATAATCTGACTAAGGCTGAACGAGGTGGCCTGTCTGAGTTGGATAAAGCCGGCTTCATCAAAAGGCAGCTTGTTGAGACACGCCAGATCACCAAGCACGTGGCCCAAATTCTCGATTCACGCATGAACACCAAGTACGATGAAAATGACAAACTGATTCGAGAGGTGAAAGTTATTACTCTGAAGTCTAAGCTGGTCTCAGATTTCAGAAAGGACTTTCAGTTTTATAAGGTGAGAGAGATCAACAATTACCACCATGCGCATGATGCCTACCTGAATGCAGTGGTAGGCACTGCACTTATCAAAAAATATCCCAAGCTTGAATCTGAATTTGTTTACGGAGACTATAAAGTGTACGATGTTAGGAAAATGATCGCAAAGTCTGAGCAGGAAATAGGCAAGGCCACCGCTAAGTACTTCTTTTACAGCAATATTATGAATTTTTTCAAGACCGAGATTACACTGGCCAATGGAGAGATTCGGAAGCGACCACTTATCGAAACAAACGGAGAAACAGGAGAAATCGTGTGGGACAAGGGTAGGGATTTCGCGACAGTCCGGAAGGTCCTGTCCATGCCGCAGGTGAACATCGTTAAAAAGACCGAAGTACAGACCGGAGGCTTCTCCAAGGAAAGTATCCTCCCGAAAAGGAACAGCGACAAGCTGATCGCACGCAAAAAAGATTGGGACCCCAAGAAATACGGCGGATTCGATTCTCCTACAGTCGCTTACAGTGTACTGGTTGTGGCCAAAGTGGAGAAAGGGAAGTCTAAAAAACTCAAAAGCGTCAAGGAACTGCTGGGCATCACAATCATGGAGCGATCAAGCTTCGAAAAAAACCCCATCGACTTTCTCGAGGCGAAAGGATATAAAGAGGTCAAAAAAGACCTCATCATTAAGCTTCCCAAGTACTCTCTCTTTGAGCTTGAAAACGGCCGGAAACGAATGCTCGCTAGTGCGGGCGAGCTGCAGAAAGGTAACGAGCTGGCACTGCCCTCTAAATACGTTAATTTCTTGTATCTGGCCAGCCACTATGAAAAGCTCAAAGGGTCTCCCGAAGATAATGAGCAGAAGCAGCTGTTCGTGGAACAACACAAACACTACCTTGATGAGATCATCGAGCAAATAAGCGAATTCTCCAAAAGAGTGATCCTCGCCGACGCTAACCTCGATAAGGTGCTTTCTGCTTACAATAAGCACAGGGATAAGCCCATCAGGGAGCAGGCAGAAAACATTATCCACTTGTTTACTCTGACCAACTTGGGCGCGCCTGCAGCCTTCAAGTACTTCGACACCACCATAGACAGAAAGCGGTACACCTCTACAAAGGAGGTCCTGGACGCCACACTGATTCATCAGTCAATTACGGGGCTCTATGAAACAAGAATCGACCTCTCTCAGCTCGGTGGAGACAGCAGGGCTGACCCCAAGAAGAAGAGGAAGGTGGAGGCCAGCGGTTCCGGACGGGCTGACGCATTGGACGATTTTGATCTGGATATGCTGGGAAGTGACGCCCTCGATGATTTTGACCTTGACATGCTTGGTTCGGATGCCCTTGATGACTTTGACCTCGACATGCTCGGCAGTGACGCCCTTGATGATTTCGACCTGGACATGCTGATTAACTCTAGAGCGGCCGCAGATCCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTAGATACGGCCGCATAG

B. Sequences of the MS2-activator constructs and corresponding gRNAbackbone vector with 2X MS2 aptamer domains is provided below (NLS,VP64, gRNA spacer, and MS2-binding RNA stem loop domains arehighlighted). Two versions of the former were constructed with theMS2_(VP64)N fusion protein format showing highest activity.

>MS2_(VP64)N (SEQ ID NO: 5)gccaccATGGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTTCTAGAATGGCTTCTAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGACTGTCGCCCCAAGCAACTTCGCTAACGGGATCGCTGAATGGATCAGCTCTAACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTCTGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGGCGCCTGGCGTTCGTACTTAAATATGGAACTAACCATTCCAATTTTCGCCACGAATTCCGACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTACGAGGCCAGCGGTTCCGGACGGGCTGACGCATTGGACGATTTTGATCTGGATATGCTGGGAAGTGACGCCCTCGATGATTTTGACCTTGACATGCTTGGTTCGGATGCCCTTGATGACTTTGACCTCGACATGCTCGGCAGTGACGCCCTTGATGATTTCGACCTGGACATGCTGATTAACTCTAGATGA >MS2_(VP64)C (SEQ ID NO: 6)gccaccATGGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTTCTAGAATGGCTTCTAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGACTGTCGCCCCAAGCAACTTCGCTAACGGGATCGCTGAATGGATCAGCTCTAACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTCTGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGGCGCCTGGCGTTCGTACTTAAATATGGAACTAACCATTCCAATTTTCGCCACGAATTCCGACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTACGAGGCCAGCGGTTCCGGACGGGCTGACGCATTGGACGATTTTGATCTGGATATGCTGGGAAGTGACGCCCTCGATGATTTTGACCTTGACATGCTTGGTTCGGATGCCCTTGATGACTTTGACCTCGACATGCTCGGCAGTGACGCCCTTGATGATTTCGACCTGGACATGCTGATTAACTCTAGAGCGGCCGCAGATCCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTAGATACGGCCGCATAG >gRNA_(2XMS2) (SEQ ID NO: 7)TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTCTGCAGGTCGACTCTAGAAAACATGAGGATCACCCATGTCTGCAGTATTCCCGGGTTCATTAGATCCTAAGGTACCTAATTGCCTAGAAAACATGAGGATCACCCATGTCTGCAGGTCGACTCTAGAAATTTTTT CTAGAC

C. dTomato fluorescence based transcriptional activation reportersequences are listed below (ISceI control-TF target, gRNA targets,minCMV promoter and FLAG tag+dTomato sequences are highlighted).

>TF Reporter 1 (SEQ ID NO: 8)TAGGGATAACAGGGTAATAGTGTCCCCTCCACCCCACAGTGGGGCGAGGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGAATTCgccaccatgGACTACAAGGATGACGACGATAAAACTTCCGGTGGCGGACTGGGTTCCACCGTGAGCAAGGGCGAGGAGGTCATCAAAGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCATGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCATGTACGGCTCCAAGGCGTACGTGAAGCACCCCGCCGACATCCCCGATTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGTCTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCACGCTGATCTACAAGGTGAAGATGCGCGGCACCAACTTCCCCCCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGATCCACCAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCAAGACCATCTACATGGCCAAGAAGCCCGTGCAACTGCCCGGCTACTACTACGTGGACACCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGCTCCGAGGGCCGCCACCACCTGTTCCTGTACGGCATGGACGAGCTGTACAAGTAA >TF Reporter 2 (SEQ ID NO: 9)TAGGGATAACAGGGTAATAGTGGGGCCACTAGGGACAGGATTGGCGAGGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGAATTCgccaccatgGACTACAAGGATGACGACGATAAAACTTCCGGTGGCGGACTGGGTTCCACCGTGAGCAAGGGCGAGGAGGTCATCAAAGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCATGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCATGTACGGCTCCAAGGCGTACGTGAAGCACCCCGCCGACATCCCCGATTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGTCTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCACGCTGATCTACAAGGTGAAGATGCGCGGCACCAACTTCCCCCCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGATCCACCAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCAAGACCATCTACATGGCCAAGAAGCCCGTGCAACTGCCCGGCTACTACTACGTGGACACCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGCTCCGAGGGCCGCCACCACCTGTTCCTGTACGGCATGGACGAGCTGTACAAGTAA

D. General format of the reporter libraries used for TALE and Cas9-gRNAspecificity assays is provided below (ISceI control-TF target, gRNA/TALEtarget site (23 bp for gRNAs and 18 bp for TALEs), minCMV promoter, RNAbarcode, and dTomato sequences are highlighted).

>Specificity Reporter Libraries (SEQ ID NO: 10)TAGGGATAACAGGGTAATAGTNNNNNNNNNNNNNNNNNNNNNNNCGAGGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGAATTCgccaccatgGACTACAAGGATGACGACGATAAANNNNNNNNNNNNNNNNNNNNNNNNACTTCCGGTGGCGGACTGGGTTCCACCGTGAGCAAGGGCGAGGAGGTCATCAAAGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCATGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCATGTACGGCTCCAAGGCGTACGTGAAGCACCCCGCCGACATCCCCGATTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGTCTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCACGCTGATCTACAAGGTGAAGATGCGCGGCACCAACTTCCCCCCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGATCCACCAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCAAGACCATCTACATGGCCAAGAAGCCCGTGCAACTGCCCGGCTACTACTACGTGGACACCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGCTCCGAGGGCCGCCACCACCTGTTCCTGTACGGCATGGACGAGCTGTAC AAGTAAGAATTC

1. A method of inserting a donor nucleic acid sequence into a cell usinghomologous recombination comprising providing to the cell two guide RNAswith each guide RNA having a spacer sequence, a tracr mate sequence anda tracr sequence, and with a portion of the tracr sequence beinghybridized to the tracr mate sequence and with the tracr mate sequenceand the tracr sequence being linked by a linker nucleic acid sequenceand with each spacer sequence being complementary to an adjacent site ina DNA target nucleic acid, providing to the cell a donor nucleic acidsequence, providing to the cell a Cas9 protein nickase, and wherein eachof the two guide RNAs co-localize with the Cas9 protein nickase to theDNA target nucleic acid resulting in an offset nick with 5′-overhangs,and wherein the donor nucleic acid sequence is inserted into the targetnucleic acid at the offset nick using homologous recombination.
 2. Themethod of claim 1 wherein the two guide RNAs are provided to the cell byintroducing into the cell a first foreign nucleic acid encoding the twoguide RNAs, wherein the Cas9 protein nickase is provided to the cell byintroducing into the cell a second foreign nucleic acid encoding theCas9 protein, and wherein the two guide RNAs and the Cas9 proteinnickase are expressed. 3-11. (canceled)
 12. The method of claim 1wherein the target nucleic acid is genomic DNA, mitochondrial DNA, viralDNA or exogenous DNA. 13-24. (canceled)
 25. The method of claim 1wherein the tracr sequence is between 90 to 500 nucleotides.
 26. Themethod of claim 1 wherein the tracr sequence is between 90 to 200nucleotides.
 27. The method of claim 1 wherein the tracr sequence isbetween 100 to 200 nucleotides.